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International Journal of Antimicrobial Agents 41 (2013) 20– 27

Contents lists available at SciVerse ScienceDirect

International Journal of Antimicrobial Agents

j our na l ho me p age: ht tp : / /www.e lsev ier .com/ locate / i jant imicag

igh in vitro antimicrobial activity of �-peptoid–peptide hybrid oligomersgainst planktonic and biofilm cultures of Staphylococcus epidermidis

ang Liua, Kolja M. Knappb, Liang Yanga,c, Søren Molina, Henrik Franzykb, Anders Folkessona,∗

Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, DenmarkDepartment of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, DenmarkSingapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore

r t i c l e i n f o

rticle history:eceived 29 June 2012ccepted 8 September 2012

eywords:eptidomimeticiofilmytotoxicitytaphylococcus

a b s t r a c t

An array of �-peptoid–peptide hybrid oligomers displaying different amino acid/peptoid compositionsand chain lengths was studied with respect to antimicrobial activity against Staphylococcus epidermidisboth in planktonic and biofilm cultures, comparing the effects with those of the common antibioticvancomycin. Susceptibility and time–kill assays were performed to investigate activity against plank-tonic cells, whilst confocal laser scanning microscopy was used to investigate the dynamics of theactivity against cells within biofilms. All tested peptidomimetics were bactericidal against both expo-nentially growing and stationary-phase S. epidermidis cells with similar killing kinetics. At the minimuminhibitory concentration (MIC), all peptidomimetics inhibited biofilm formation, whilst peptidomimeticsat concentrations above the MIC (80–160 �g/mL) eradicated young (6-h-old) biofilms, whilst even higherconcentrations were needed to eradicate mature (24-h-old) biofilms completely. Chiral and guanidiny-lated hybrids exhibited the fastest killing effects against slow-growing cells and had more favourable

antibiofilm properties than analogues only containing lysine or lacking chirality in the �-peptoid residues.However, the results of the mature biofilm killing assay indicated more complex structure–activity rela-tionships. Cytotoxicity assays showed a clear correlation between oligomer length and cell toxicity withineach subclass of peptides, but all possessed a high differential toxicity favouring killing of bacterial cells.This class of peptidomimetics may constitute promising antimicrobial alternatives for the preventionand treatment of multidrug-resistant S. epidermidis infections.

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© 2012 E

. Introduction

Infections with the opportunistic human pathogen Staphy-ococcus epidermidis frequently affect immunocompromised andmmunosuppressed patients, especially when subjected to pro-onged use of indwelling medical devices [1]. The prevalentntibiotic resistance and ability to form biofilms have led to anncrease in complications associated with the treatment of S. epi-ermidis infections [2]. Staphylococcus epidermidis biofilms on theurface of indwelling devices such as catheters and prosthetic heartalves are difficult to treat with conventional antibiotics [3]. Thencreased antibiotic tolerance of biofilm-associated cells has beenorrelated with their slow growth rate, protection mediated byxtracellular polymeric substances and the development of tol-

rant subpopulations [1,4]. Discovery and development of novelgents for the prevention and treatment of S. epidermidis biofilmnfections are therefore urgently needed.

∗ Corresponding author. Tel.: +45 4525 2767.E-mail address: anf@bio.dtu.dk (A. Folkesson).

924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemttp://dx.doi.org/10.1016/j.ijantimicag.2012.09.014

r B.V. and the International Society of Chemotherapy. All rights reserved.

Natural host defence antimicrobial peptides (AMPs) are pro-duced by most living organisms and, owing to their uniqueproperties and alternative modes of action, they are consideredto be effective against multidrug-resistant bacteria [5]. AMPs havealso been reported to efficiently kill slow-growing cells from plank-tonic and biofilm cultures and thus they have been proposedas promising alternative agents in the treatment of multidrug-resistant infections [6]. Peptidomimetics are structural analoguesof peptides containing amide bond isosteres or altered peptidebackbones that result in higher stability as well as improved phar-macological profiles. Typically, peptidomimetics arise either frommodification of an existing active peptide or from the designof structurally similar compounds that mimic peptides, e.g. �-peptides or peptoids. Appropriately designed peptidomimeticshave been shown to be capable of maintaining a broad spectrum ofantimicrobial activity whilst possessing advantageous propertiesover natural AMPs, such as stability against proteolytic enzymes

and low toxicity towards mammalian cells [7].

Previously we have described a synthetic approach for thedesign of peptidomimetics consisting of alternating repeats of�-amino acids and �-peptoid residues [8–11]. These studies

otherapy. All rights reserved.

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uggested that one strategy to design peptidomimetics with aavourable balance between potency and cytotoxicity involvesncorporation of chiral hydrophobic �-peptoids and guanidinylatedmino acid side chains whilst keeping the length relatively short.

In the present study we investigated the antimicrobialctivity of the simple alternating �-peptoid–peptide hybridligomers (i.e. 1a–3d) and the mixed amino/guanidino subtypef peptidomimetics (i.e. 4a–4d) against the biofilm-forming andethicillin-resistant S. epidermidis (MRSE) strain RP62A (ATCC

5984) both in planktonic and biofilm cultures.Their effect is compared with that of vancomycin (VAN), which

s commonly used for the treatment of resistant or severe Gram-ositive organisms, and thus constitutes the current antibiotic of

last resort’ [12].

. Materials and methods

.1. Synthesis of ˇ-peptoid–peptide hybrid oligomers

The four mixed amino- and guanidino-functionalised oligomersa–d [13] were synthesised using our previously described solid-hase procedure [8]. The �-peptoid–peptide hybrid oligomersere dissolved in sterile deionised water (5 mg/mL) and aliquotsere stored at −20 ◦C.

.2. Haemolysis assay

Haemolysis was performed as described previously usinguman erythrocytes [14], detecting haemoglobin by measuring theptical density at 405 nm. Melittin (400 �g/mL) and Tris buffer (pH.2, 150 mM NaCl) defined 100% haemolysis and 0% haemolysis,espectively.

.3. Cytotoxicity assay

The cytotoxicity assay was performed essentially as reportedreviously using HeLa cells [11]. Briefly, HeLa cells were incu-ated at 37 ◦C for 1 h with peptidomimetics. Incubation waserformed using a horizontal shaking table (with a frequency of 50ovements/min) that was placed in a custom-made temperature-

ontrolled polystyrene cabinet. The tested concentration range was.1–1000 �M. Dehydrogenase activity was determined as a resultf the amount of formazan produced as measured by absorbancet 492 nm.

.4. Bacterial strains and growth media

Staphylococcus epidermidis RP62A (ATCC 35984) strain waselected as the model organism for this study as it is considered

benchmark strain among the biofilm-producing S. epidermidistrains [15]. Tryptic soy broth (TSB) (Oxoid Ltd., Basingstoke, UK)ontaining 0.25% glucose was used for biofilm cultivation in atatic chamber system. Susceptibility assays were carried out inueller–Hinton broth (MHB) (Fluka, Steinheim, Germany). SYTO

and propidium iodide (LIVE/DEAD® reagents; Molecular Probes,ugene, OR) were used at a concentration of 1 �M for staining liver dead bacteria in biofilms.

.5. Bacterial susceptibility assay

Minimum inhibitory concentrations (MICs) were measured

sing methods described previously by Wiegand et al. [16]. Theinimum bactericidal concentration (MBC) was determined as the

owest concentration that resulted in <0.1% survival of the sub-ulture. All MIC and MBC determinations were made in triplicate.

microbial Agents 41 (2013) 20– 27 21

For selected compounds, MICs were also measured using the samemethod in the biofilm medium (TSB with 0.25% glucose).

2.6. Time–kill assay in fresh Mueller–Hinton broth

Staphylococcus epidermidis cells (1 × 107 CFU/mL) were sepa-rately treated with �-peptoid–peptide hybrid oligomers at 4 �g/mLor VAN at 4 �g/mL and 20 �g/mL in fresh MHB. Time–kill exper-iments were performed at 37 ◦C with shaking at 220 rpm underaerobic conditions. Culture aliquots (50 �L) were taken at differ-ent time points (0, 1, 3, 5, 8 and 24 h), serially diluted, plated ontotryptic soy agar and then incubated at 37 ◦C for 24 h followed bycolony counting. Time–kill curves were constructed by plottingthe log10 CFU/mL versus time over a 24-h time period. Assayswere performed in duplicate on at least two occasions and simi-lar results were obtained. The detection limit for these assays was5 × 102 CFU/mL.

2.7. Time–kill assay of stationary-phase cells in nutrient-depletedMueller–Hinton broth

Nutrient-depleted (or spent) MHB (depMHB) was used toarrest cell growth and to keep cells in the slow-growing sta-tionary phase. depMHB was prepared by the method describedpreviously with modifications [17]. Briefly, S. epidermidis was cul-tivated in MHB at 37 ◦C for 46 h. Cultures were centrifuged at8000 × g for 30 min at 4 ◦C and the supernatants were collectedand adjusted to pH 7.0, then filtered through a 0.22 �m pore sizesyringe filter (TPP, Trasadingen, Switzerland). Stationary-phasecells (1 × 107 CFU/mL) were separately incubated in depMHB in thepresence of �-peptoid–peptide hybrid oligomers or VAN at 4 �g/mLand 20 �g/mL. The time–kill assay was performed as above.

2.8. Biofilm susceptibility assay

Static chamber S. epidermidis biofilms were cultivated in a cover-glass cell culture chamber (Nunc, Roskilde, Denmark) as describedpreviously [18]. Briefly, the chamber wells were inoculated with1.5 mL of diluted overnight cultures (5 × 105 CFU/mL). Followingincubation at 37 ◦C, the 0-, 6- and 24-h pre-formed biofilms inthe chambers were washed gently twice with sterile phosphate-buffered saline (1 mL) to remove planktonic cells. Fresh mediumcontaining antimicrobial agents was then added and the biofilmcultures were incubated at 37 ◦C for 24 h. After removal of themedium, biofilm cells were stained with the LIVE/DEAD reagentsand were then observed by confocal laser scanning microscopy(CLSM). This assay was repeated three times and similar resultswere obtained.

2.9. Microscopy image acquisition and analysis

All microscopy observations and image acquisitions were per-formed using methods described previously by Qin et al. [18].For quantification of biofilms, at least six CLSM images from eachsample were analysed using the computer program COMSTAT. Sta-tistical differences in comparison with the control (without addedantimicrobial agent) were determined by one-way analysis of vari-ance (ANOVA). Differences were considered statistically significantat a P-value of <0.05.

3. Results

In the present study, 16 �-peptoid–peptide hybrid oligomers(1a–4d) (Table 1; Fig. 1) were investigated for their antimicro-bial activity against both planktonic and biofilm-associated S.epidermidis cells as well as for their cytotoxicity towards human

22 Y. Liu et al. / International Journal of Antimicrobial Agents 41 (2013) 20– 27

Table 1Antimicrobial, haemolytic and cytotoxic activities of peptidomimetics.

Subclass Compound no. Mw (g/mol) MIC (�g/mL) [�M]a HC10 for hRBCs (�g/mL)a,d IC50 for HeLa cells (�g/mL) [�M]a

MHBb Biofilm mediumc

1: all-amino; �-chiral 1a 1624.81 32 [20] >128 [>79] >500 N/D1b 1937.74 4 [2.1] >128 [>66] >500 >1938 [>1000]1c 2250.67 2 [0.9] 64 [28] >500 N/D1d 2563.60 2 [0.8] 16 [4.8] >500 92 [36]

2: all-guanidino;�-chiral

2a 2356.38 2 [0.9] N/D >500 N/D2b 2815.84 1 [0.4] 4 [1.4] >500 90 [32]2c 3275.30 2 [0.6] 8 [2.4] >500 N/D2d 3734.76 4 [1.1] N/D >500 27 [7.2]

3: all-guanidino;no �-chirality

3a 2286.25 4 [1.8] 32 [14] >500 N/D3b 2732.69 1 [0.4] 8 [2.9] >500 262 [96]3c 3179.13 2 [0.6] 16 [5.0] >500 N/D3d 3625.57 2 [0.6] 16 [4.4] >500 40 [11]

4: amino-guanidino1:1; �-chiral

4a 935.95 >256 [>273] N/D N/D N/D4b 1812.84 32 [18] >128 [>71] N/D N/D4c 2689.72 2 [0.7] 16 [6.0] >512 794 [295]4d 3566.61 1 [0.3] 4 [1.1] 512 46 [13]

Vancomycin 1485.71 1 [0.7] 2 [1.4] N/D N/D

Mw, molecular weight; MIC, minimum inhibitory concentration; HC10, concentration that causes 10% haemolysis; hRBCs, human red blood cells; IC50, 50% maximuminhibitory concentration against HeLa cells; N/D, not determined.

a MIC, HC10 and IC50 values represent the mean of three individual experiments.b MIC towards planktonic cultures of Staphylococcus epidermidis in Mueller–Hinton broth.c MIC towards planktonic cultures of S. epidermidis in biofilm medium (tryptic soy broth with 0.25% glucose).

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d Values of compounds belonging to subclasses 1–3 were taken from the literatu

ells. Peptidomimetics 1a–4d are oligomers consisting of alternat-ng repeats of �-amino acids and N-alkyl-�-alanine (�-peptoid)esidues. The four examined subclasses differ in the nature ofationic side chains [amino- or guanidino-functionalised, i.e. Lysr homoarginine (hArg)] and presence of �-chirality in the �-eptoid residues (�Nspe or �Nphe). The representative compoundsf the latter subclass (4) have chain lengths corresponding to 4–16esidues.

.1. Haemolytic and cytotoxic properties of ˇ-peptoid–peptideybrids

Peptidomimetics 1b, 1d, 2b, 2d, 3b and 3d had previously beenound to exhibit minimal haemolysis (<10%) even at a concentrationf 500 �g/mL, and similarly low haemolytic activity was deter-ined in the present work for compounds 4c and 4d (at 512 �g/mL,

ig. 1. Chemical structures of the �-peptoid–peptide hybrids. Abbreviations used for the-prefix: �Nspe, N-[(S)-1-phenylethyl]-�-alanine; �Nphe, N-phenyl-�-alanine; hArg, ho

].

<5% and 10% haemolysis, respectively) by using the same conditions[11].

From each subclass of peptidomimetics, the oligomers consist-ing of 12 or 16 residues were investigated for their cytotoxicityagainst human HeLa cells (Table 1), and the sequence length andcontent of guanidinium side chains clearly turned out to be themost significant structural features influencing this pharmacolog-ical property. Thus, potency in killing human cells increased ca.20-fold when extending the sequences from 12 to 16 residueswithin the Lys-containing subclasses (1 and 4), whereas this effectwas less pronounced (3–7-fold increase) for the all-hArg analogues(i.e. 2b, 2d, 3b and 3d). Noticeably, all 16-meric peptidomimet-ics exhibited relatively high cytotoxicity [50% maximal inhibitory

concentration (IC50) � 100 �M] compared with the 12-mers con-taining lysine (i.e. 1b and 4c). Similarly, the lack of �-chirality in the�-peptoid residues in 3b was accompanied by a 3-fold diminishedcytotoxicity relative to the corresponding fully chiral oligomer (2b).

�-peptoid units were adapted from those used for �-peptoids [19] by adding themoarginine.

of Antimicrobial Agents 41 (2013) 20– 27 23

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Fig. 2. Time–kill curves for Staphylococcus epidermidis RP62A (ATCC 35984) uponexposure to �-peptoid–peptide hybrids and vancomycin (VAN). Cells were treatedwith compounds at low and high concentrations (4 �g/mL and 20 �g/mL) indepleted Mueller–Hinton broth (MHB) (B and C) as well as at a low concentration of

Y. Liu et al. / International Journal

.2. Susceptibility testing in Mueller–Hinton broth and biofilmedium

The concentrations of �-peptoid–peptide hybrid oligomersequired for inhibition or killing of planktonic S. epidermidis cellsn MHB or biofilm medium (TSB with 0.25% glucose) are summ-rised in Table 1. For subclasses 1 and 4, MIC values decreasedignificantly with increasing oligomer length. Thus, chimeras 1dnd 4d ranked the most potent in their subclasses, with MICs of

�g/mL and 1 �g/mL, respectively. In contrast, less pronouncedifferences in MICs were found for members among subclasses 2nd 3, which all were active in the range of 1–4 �g/mL. Hybrids 2bnd 3b, which are dodecamers, proved most effective within theirubclass, both exhibiting MICs of 1 �g/mL. The MIC of the refer-nce antibiotic VAN was 1 �g/mL. MBCs of the compounds werell equal to or double the MICs, clearly indicating a bactericidalode of action. However, it was found that S. epidermidis cells were

ess susceptible to the peptidomimetics when challenged in biofilmedium, where 2b and 4d exhibited the lowest MICs (4 �g/mL),hilst the MIC of VAN was 2 �g/mL. The peptidomimetics 1d, 2b,

b, 4c and 4d all exhibited high antimicrobial activity and selectiv-ty (i.e. IC50 � MIC; Table 1) in the killing of bacteria over humanells, and therefore these five chimeras were further investigated.

.3. ˇ-Peptoid–peptide hybrids efficiently kill fast-growingtaphylococcus epidermidis cells

Time–kill kinetics was investigated in fresh MHB to deter-ine whether �-peptoid–peptide hybrid oligomers and VAN are

ffective against fast-growing S. epidermidis cells (Fig. 2). Five-peptoid–peptide hybrid oligomers (1d, 2b, 3b, 4c and 4d) rep-

esenting all subclasses as well as VAN at a concentration of �g/mL (equal to 2–4 times their MICs) were separately addedo the cultures in order to determine their effect on cell viabil-ty compared with untreated control cells. The starting inoculum

as 1 × 107 CFU/mL. Untreated control cells grew fast (increasinga. 2 log10 CFU/mL over a 24-h period). Compound 4d (4 �g/mL)xhibited the fastest bactericidal activity in growing cultures of S.pidermidis as it produced a 3 log reduction in CFU/mL within 1 h.ligomers 1d and 2b required a longer time (3 h) to achieve a sim-

lar bactericidal effect, whilst compounds 3b and 4c needed 5 h tochieve a 3 log reduction in CFU/mL. Notably, VAN exhibited a morerotracted bactericidal activity than the five peptidomimetics as5 h were required to achieve a bactericidal effect. Increasing theAN concentration to 20 �g/mL did not improve the killing curve,hich suggests that it has a concentration-independent activity

gainst fast-growing S. epidermidis cells.

.4. ˇ-Peptoid–peptide hybrids efficiently kill slow-growingtationary-phase Staphylococcus epidermidis cells

The potency of the peptidomimetics was determined againstlow-growing stationary-phase S. epidermidis cultures in depMHB.tationary-phase S. epidermidis cultures were left untreated orere treated separately with compounds 1d, 2b, 3b, 4c, 4d orAN at 4 �g/mL (Fig. 2B). It took ca. 3 h for compound 4d, 6 h forompounds 2b and 1d and 24 h for compound 4c to produce a bac-ericidal effect (Fig. 2B). Oligomer 3b caused a ca. 2 log decrease iniability after 24 h, whereas VAN (4 �g/mL) did not exhibit a killingffect under these growth conditions.

Moreover, to determine whether the killing effects areoncentration-dependent, stationary-phase S. epidermidis cultures

ere left untreated or were treated separately with the compounds

t 20 �g/mL, which corresponds to ca. 10 times the MIC concentra-ion (Fig. 2C). Compounds 2b and 4d exhibited rapid bactericidalctivities with a >3 log reduction after 1 h, whilst compounds 1d,

�-peptoid–peptide hybrid oligomers (4 �g/mL) or VAN at 4 �g/mL and 20 �g/mL infresh MHB (A). Viability was counted at the indicated time points by serial dilutionplating. Values are the mean of independent tests performed in duplicate.

4c and 3b gave bactericidal effects after 3, 8 and 24 h, respectively.Notably, VAN at high concentration (20 �g/mL) caused a decreaseof ca. 2 log after 24 h.

3.5. ˇ-Peptoid–peptide hybrids inhibit Staphylococcusepidermidis biofilm formation

Next, the potential of �-peptoid–peptide hybrid oligomers as

prophylactic agents for preventing S. epidermidis biofilm forma-tion was investigated. Thus, the selected hybrids were tested at lowconcentration (≤1× MIC) in an assay allowing for quantification ofbiofilm formation using fluorescence microscopy and LIVE/DEAD

24 Y. Liu et al. / International Journal of Antimicrobial Agents 41 (2013) 20– 27

Fig. 3. Effects of antimicrobials in preventing formation of Staphylococcus epidermidis RP62A (ATCC 35984) biofilms when tested at sub-minimum inhibitory concentrations(sub-MIC) and at 1× MIC. (A) Confocal laser scanning microscopy (CLSM) images. Biofilms were stained with SYTO 9 (green fluorescent for live cells) and propidium iodide(red fluorescent for dead cells). The central pictures show horizontal CLSM optical sections, and the flanking pictures show vertical CLSM optical sections. Bars represent2 STAT a* symbb

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0 �m. Assays were repeated three times and similar results were obtained. (B) COM**,###P < 0.001) with respect to the control (no antimicrobial added); absence of aars represent the mean + standard deviation. VAN, vancomycin.

taining (Fig. 3). At the MICs, all five peptidomimetics as well asAN prevented formation of S. epidermidis biofilms. In particular,ompound 2b (4 �g/mL) inhibited biofilm formation, correspond-ng to a 100% reduction of biomass. Furthermore, whether sub-MICs1–2 �g/mL) of such peptidomimetics were able to prevent biofilmormation was tested. For oligomers 1d and 4c, a concentration of

�g/mL was used due to their relatively high MICs (16 �g/mL). Athese sub-MICs, oligomers 4d (1 �g/mL) and 1d (2 �g/mL) causedignificant reductions of biomass (ca. 65% and 40%, respectively),hilst VAN (1 �g/mL) caused a 65% reduction. However, the killing

ffect of VAN appeared to be very heterogeneous since some partsf the biofilm were reduced whilst other parts were not. Conse-uently, the remaining amount of biomass at different positions ofhe static chamber varied greatly, as reflected in a large standardeviation (Fig. 3).

.6. Activity of ˇ-peptoid–peptide hybrids towards youngtaphylococcus epidermidis biofilms

To determine whether the selected peptidomimetics were effec-ive also for killing of bacterial cells within biofilms, CLSM inombination with SYTO 9 DNA viability staining were used to eval-ate the produced amount of biomass as a measure of cell viability.ifferences in bacterial survival in the biofilms were quantified bynalysing CLSM data with the computer program COMSTAT [20].or the young (6-h) S. epidermidis biofilms, the peptidomimetics

ere added at low and high concentrations (Fig. 4). At low concen-

rations (1–4× MIC; 8–16 �g/mL), peptidomimetics 1d, 2b, 4c andd all reduced cell viability by 80–85%, whereas oligomer 3b had noignificant effect. The reference antibiotic VAN (8 �g/mL) gave rise

nalysis of biomass. Symbols indicate a statistically significant difference (**P < 0.01;ol indicates no statistically significant difference with respect to the control. Error

to ca. 50% reduction in live biomass, however the killing effects ofboth VAN and oligomer 3b appeared to be very heterogeneouslydistributed within the biofilm. Nevertheless, at high concentra-tions (10–40× MIC; 80–160 �g/mL), all tested compounds almostcompletely disrupted pre-formed S. epidermidis biofilms (95–100%reduction of biomass) (Fig. 4).

3.7. Activity of ˇ-peptoid–peptide hybrids towards matureStaphylococcus epidermidis biofilms

In the study of the killing effect towards mature (24-h-old) S.epidermidis biofilms, the selected peptidomimetics were tested atthree different concentration levels (Fig. 5). At low concentrations(8–16 �g/mL), only oligomer 4d (8 �g/mL) was able to cause ca. 43%reduction of viable biomass. At a high concentration (160 �g/mL),oligomers 4d and 4c as well as VAN reduced live biomass by 71%,46% and 50%, respectively. At 400 �g/mL, peptidomimetics 4d,2b and 4c as well as VAN caused significant reductions in livingbiomass (78%, 77%, 45% and 69%, respectively). Hybrid oligomers 4dand 2b were most potent in killing of mature biofilm in accordancewith their low MICs in biofilm medium and high activities againststationary-phase S. epidermidis cells (Table 1; Fig. 3). Althoughless effective than oligomer 1d in the time–kill study, oligomer 4cexhibited a better killing effect on mature biofilm than compound1d. Surprisingly, although VAN was not as potent as the testedpeptidomimetics in killing slow-growing stationary-phase cells,

it exhibited significant activity against cells in mature biofilms.The observed distinct heterogeneity of survival in the VAN-treatedbiofilms suggests that the mode of action for VAN is different fromthat of the peptidomimetics.

Y. Liu et al. / International Journal of Antimicrobial Agents 41 (2013) 20– 27 25

Fig. 4. Effects of antimicrobials against established Staphylococcus epidermidis RP62A (ATCC 35984) young (6-h-old) biofilm when tested at 8–16 �g/mL and 80–160 �g/mL.(A) Confocal laser scanning microscopy images. Bars represent 20 �m. (B) COMSTAT analysis of live biomass. Symbols indicate a statistically significant difference (*P < 0.05;* ,###

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** P < 0.001) with respect to the control (no antimicrobial added); absence of aars represent the mean + standard deviation. VAN, vancomycin; AMP, antimicrobi

. Discussion

This study set out to probe the relationship between structuresequence composition and chain length) and activities (antimicro-ial, antibiofilm and cytotoxic) for a series of �-peptoid–peptideybrid oligomers using the biofilm-forming and methicillin-esistant strain S. epidermidis RP62A as a model organism.

The susceptibility of S. epidermidis planktonic cells exhibitedimilar trends in structure–activity relationships as found in pre-ious studies. Thus, a longer chain length was often correlatedith increased antimicrobial activity within each subclass. This

endency was more pronounced in lysine-containing subclasses (1nd 4) than in homoarginine-rich subclasses (2 and 3) (Table 1).

Staphylococcus epidermidis cells were found to be less sus-eptible to the peptidomimetics when tested in biofilm mediumTSB + 0.25% glucose) compared with MHB (Fig. 2; Table 1). Thisifference is most likely due to the fact that S. epidermidis growsaster and reaches a higher cell density in biofilm medium than in

HB (data not shown).In the present study, peptidomimetics 1b and 4c were found

o possess the most favourable cytotoxicity profiles towards HeLaells (IC50 > 1900 �g/mL and 794 �g/mL, respectively), suggestinghat a design of alternating 12-meric oligomers displaying onlymino or guanidino/amino functional groups in a 1:1 ratio maye a promising strategy to keep cytotoxicity at an acceptable level.owever, the all-Lys compound 1b did not show any significantntimicrobial activity in biofilm medium, indicating that some con-ent of guanidino side chains is required for antibiofilm activity.ikewise, the haemolytic activity of 4c proved favourably low as noaemolysis was detected at a concentration of 256 �g/mL and <5%aemolysis was seen at 512 �g/mL.

The finding that hArg-rich peptidomimetics belonging to sub-lasses 2 and 3 exhibited significant cytotoxicity towards humanells may be ascribed to their improved interaction with humanembranes promoted by the ability of guanidinium groups to form

ol indicates no statistically significant difference with respect to the control. Errortide.

bidentate hydrogen bonds with the phospholipids that constitutethe major part of eukaryotic membranes.

Chirality appears essential for efficient killing of both slow-growing planktonic cells and biofilm cultures, which corroboratespreliminary studies showing that the presence of chiral �-peptoidresidues results in higher antimicrobial potency against Staphylo-coccus aureus, which may be due to the higher degree of secondarystructure in compounds 1d/2b versus compound 3b. In comparisonwith their lysine-containing counterparts (e.g. 1d and 4c), the fullyguanidinylated (i.e. hArg-rich) oligomer (e.g. 2b) exhibited fasterkilling of slow-growing cells and possessed enhanced antibiofilmcapacity in accordance with our previous studies showing thatthe presence of hArg residues in this type of peptidomimeticsstrongly promotes activity against S. aureus. There was little cor-relation between the killing ability towards slow-growing cellscompared with mature biofilms. Hybrid 4c was much less potenttowards slow-growing cells compared with compound 2b, how-ever 4c exhibited higher killing activity towards mature biofilmcells than 2b.

In particular, there is a need for studies aimed at elucidatingthe mode of action of these peptidomimetics against S. epidermidisas well as estimating the potential resistance mechanisms of S.epidermidis planktonic and biofilm cells against these oligomers.The efficient killing of slow-growing bacterial cells exerted bothby AMPs and antimicrobial peptidomimetics may be due to theirdistinct killing mechanisms. Unlike conventional antibiotics thatpenetrate into bacterial cells and interfere with intracellular tar-gets, most AMPs and antimicrobial peptidomimetics appear to actmainly via pore formation or mechanisms involving transient dis-ruption of the integrity of bacterial cell membranes [21]. However,there is growing evidence that several highly potent AMPs may in

fact exert their antibacterial effect via intracellular targets [22,23].

The tolerance of S. epidermidis biofilm cells towards �-peptoid–peptide hybrid oligomers may be due to the inductionof the AMP-sensing (Aps) regulatory system [1], which is well

26 Y. Liu et al. / International Journal of Antimicrobial Agents 41 (2013) 20– 27

F A (ATC nalysi* absenc , anti

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bVmoitsp�b

ig. 5. Effects of antimicrobials against established Staphylococcus epidermidis RP62onfocal laser scanning microscopy images. Bars represent 20 �m. (B) COMSTAT a*P < 0.01; ###,∼∼∼P < 0.001) with respect to the control (no antimicrobial added);

ontrol. Error bars represent the mean + standard deviation. VAN, vancomycin; AMP

onserved among staphylococci. In S. aureus, the Aps system acti-ates transcription of three genomic sequences involved in AMPesistance, including mprF, the dlt operon and vraFG [1]. The S.ureus mprF gene encodes LPG synthetase that transfers l-lysinerom lysyl-tRNA to phosphatidylglycerol (PG) [24]. The S. aureus dltperon is responsible for the d-alanylation of teichoic acid, whichs a highly negatively charged component of the cell wall poly-

er lipoteichoic acid (LTA) [25]. The aminoacylation of PG andTA results in partial neutralisation of the negative charge of the S.ureus cell envelope and thereby reduces its susceptibility towardsMPs [26]. The vraFG operon encodes an ABC transporter involved

n the resistance of S. aureus to AMPs and VAN. These resistanceystems of S. aureus have homologues in S. epidermidis, howeverurther studies need to be performed to establish whether theyre involved in the tolerance of S. epidermidis cells towards �-eptoid–peptide hybrid oligomers [1].

The results obtained for the treatment of mature S. epidermidisiofilms with selected �-peptoid–peptide hybrid oligomers andAN indicate the presence of a physiological differentiation inature S. epidermidis biofilms conferring overall increased antibi-

tic tolerance to the cell cultures (Fig. 5). The slow-growing cellsn the biofilm are not only more tolerant towards VAN but alsoowards the peptidomimetics. This tolerance might be due to the

ubstantial amount of extracellular polymeric substances such asoly-N-acetylglucosamine (PNAG, also known as PIA) and poly--glutamic acid (PGA) present in surface layers of S. epidermidisiofilms [27]. Both PNAG and PGA protect S. epidermidis from

CC 35984) mature (24-h-old) biofilm when tested at 8–16, 160 and 400 �g/mL. (A)s of live biomass. Symbols indicate a statistically significant difference (#,∼P < 0.05;ce of a symbol indicates no statistically significant difference with respect to themicrobial peptide.

AMPs and phagocytosis [27]. However, resistant S. epidermidis cellswere found to be distributed randomly upon treatment with pep-tidomimetics, suggesting that other mechanisms might be involvedin the tolerance observed for �-peptoid–peptide hybrid oligomers.

These experiments clearly demonstrate that the present class ofpeptidomimetics is able to kill both fast-growing and slow-growingbacterial cells. Furthermore, the tested hybrid oligomers inhibitedformation of S. epidermidis biofilms and displayed antibiofilm activ-ity against pre-formed S. epidermidis biofilms. Also, the combinedstructure–activity relationships regarding their overall pharma-cological profiles (i.e. antimicrobial activity versus cytotoxicity)indicate that these peptidomimetics constitute promising leadcompounds towards S. epidermidis biofilms owing to their highantibiofilm activity combined with low cytotoxicity and negligiblehaemolytic activity. We have shown that these peptidomimeticspossess bactericidal activity that is comparable, if not higher than,the current ‘last resort’ antibiotic VAN. This class of compoundsshould therefore be considered relevant alternatives in the searchfor novel treatment options for severe nosocomial Gram-positiveinfections.

Funding: YL was funded by a PhD grant from the Technical Uni-versity of Denmark and the Danish Research Council for Technologyand Production (grant no. 09-065902/FTP). The authors thank the

Brødrene Hartmanns Fond (Copenhagen, Denmark) for a materialsgrant supporting the synthesis work.

Competing interests: None declared.Ethical approval: Not required.

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