Screening for a potent antibacterial peptide to treat

mupirocin-resistant MRSA skin infections.

Siew Mei Samantha Ng1^, Hui Si Vivian Ching1^, GuiFang Xu1^, Fui Mee Ng1, Esther H. Q. Ong1, Qiu

Ying Lau1, Roland Jureen2, Jeffrey Hill1 and C. S. Brian Chia1*

1 Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31

Biopolis Way, Nanos #03-01, Singapore 138669.

2 Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road 119074,

Singapore

^ These authors contributed equally to this work.

* Corresponding author.

Tel.: +65 64070348

E-mail: cschia@etc.a-star.edu.sg (Brian Chia)

Key words: antimicrobial peptide, mupirocin-resistant, MRSA, skin infection, nasal decolonization

1

Abstract

Mupirocin is the first-line topical antibacterial drug for treating skin infections caused primarily by

meticillin-resistant Staphylococcus aureus (MRSA). Its widespread use since its introduction more

than 30 years ago has resulted in the global emergence of mupirocin-resistant strains of MRSA.

Antimicrobial peptides (AMPs) are a promising class of antibacterial compounds that can potentially

be developed to replace mupirocin due to their rapid membrane-targeting bactericidal mode of action

and predicted low propensity for resistance development. Herein, we conducted and compared the

antibacterial activities of 61 AMPs between 3 to 11 residues in length reported in the literature over

the past decade against mupirocin-resistant MRSA. The most potent AMP, 11-residue peptide 50,

was selected and tested against a panel of clinical isolates followed by a time-kill and a human

dermal keratinocyte cytotoxicity assay. Lastly, peptide 50 was formulated into a topical spray which

showed strong in vitro bactericidal effects against mupirocin-resistant MRSA. Our results strongly

suggest that peptide 50 has the potential to be further developed into a new class of topical

antibacterial agent for treating drug-resistant MRSA skin infections.

2

1. Introduction

Staphylococcus aureus is the primary pathogen responsible for the majority of bacterial skin and soft

tissue infections (SSTIs), representing more than 75% of cases in a 2004 survey conducted in 11 US

hospitals (Dryden 2010; Moran et al 2006). Of these, almost 60% were caused by meticillin-resistant

S. aureus (MRSA) (Moran et al 2006). Indeed, MRSA has become endemic in many hospitals

worldwide, posing a huge medical and economic burden to the healthcare industry (Gould 2006) and

prompting the US Centers for Disease Control and Prevention (CDC) to declare MRSA as a serious

threat in their 2013 antibiotic resistance threat report (Frieden 2013).

The first-line topical antibacterial drug prescribed for treating MRSA skin infections, Mupirocin, was

first introduced in 1985 (Eltringham 1997; Stevens et al 2005; Upton et al 2003). Its long history and

widespread use has inevitably lead to the emergence of mupirocin-resistant MRSA strains worldwide

(Antonov et al 2015; Hetem et al 2013; Lepainteur et al 2013; Park et al 2012; Patel et al 2009; Upton

et al 2003; Youn et al 2015). For example, a 2012 survey involving 7 public hospitals in Singapore

revealed approximately 31% of MRSA isolated from patients were mupirocin-resistant (Hon et al

2014), mirroring results from a 2013 survey in New York (Antonov et al 2015). Such alarming

statistics necessitates the development of new classes of topical antibacterials to replace mupirocin

(Coates et al 2009; Poovelikunnel et al 2015). The problem is further exacerbated by the paucity of

new classes of antibiotics entering the market due to the perceived low investment returns compared

to drugs for chronic diseases (Kinch et al 2014).

Antimicrobial peptides (AMPs) have generated significant interest as antibacterials (Yeung et al 2011;

Zasloff 2002). AMPs can be designed to specifically target bacteria membranes, exerting their

bactericidal effects by membrane insertion and disruption, resulting in cell lysis (Yeung et al 2011;

Zasloff 2002). This rapid mode of action reduces the chances of bacteria acquiring resistance,

imbibing them with a significant advantage over traditional antibiotics which target bacterial proteins

or cell-wall intermediates (Yeung et al 2011; Zasloff 2002). However, AMPs suffer two major

disadvantages as drug candidates: they are susceptible to proteolytic degradation due to endogenous

human proteases and are perceived to be expensive to manufacture due to their relatively large sizes

compared to conventional antibiotics (Brogden and Brogden 2011; Hancock and Sahl 2006; Marr et

3

al 2006; Zhang and Fella 2006). The first drawback can be addressed by formulating AMPs for topical

applications to treat bacterial skin infections and the second disadvantage can be addressed by

designing very short AMPs with high selectivity for bacteria with potent antibacterial properties.

Indeed, the 12-residue AMP, Omiganan, is currently in phase 2 clinical trials for treating

Propionibacterium acnes, the bacterium responsible for acne (ClinicalTrials.gov identifier:

NCT02571998) (Sader et al 2004). Herein, we report our effort to identify a shorter but more potent

AMP to gauge its potential for further drug development as a topical antibacterial for treating SSTIs

caused by mupirocin-resistant MRSA. Towards this, we started off by conducting a PubMed search

for AMPs between 2 to 11 residues reported over the last decade (2006 - 2015) with antibacterial

activities. To minimize manufacturing costs, only peptides with natural amino acids were selected.

These AMPs were then synthesized commercially and subjected to a head-to-head comparison

based on their minimum inhibitory concentrations (MICs) against mupirocin-resistant MRSA. 8 FDA-

approved topical antibacterials and the AMP clinical candidate Omiganan were included for

comparison. The most potent peptide was then screened against a panel of clinical MRSA isolates

followed by a bactericidal/static determination assay, a time-kill assay and a toxicity study using

normal human epidermal keratinocytes. Lastly, the peptide candidate was formulated into a spray and

subjected to an in vitro MRSA growth inhibition assay to gauge its potential for further development as

a topical antibacterial to treat MRSA skin infections.

2. Material and Methods

Bacitracin zinc, clindamycin hydrochloride, erythromycin, melittin, Mueller Hinton 2 (MH2) agar

powder, mupirocin calcium, neomycin trisulfate, polymyxin B sulfate, retapamulin, tetracycline

hydrochloride and propylene glycol were purchased from Sigma-Aldrich (USA) and were used without

further purification. All peptides reported in this study were synthesized by Mimotopes (Australia) to

>95% purity. Ethanol (99.9%) was purchased from Merck KGaA (Germany). Cation-adjusted Mueller

Hinton broth 2 (MH2) was purchased from BD (USA). All bacteria were purchased from ATCC (USA)

with the exception of MRSA NUH-K751, a clinical isolate obtained from the National University

Hospital, Singapore. Disposable travel spray bottles (20 mL; model 40-839; www.selwapro.com) were

purchased from 2WO Pte Ltd (Singapore).

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2.1 PubMed search criteria

Search terms 'antimicrobial peptides' and 'antibacterial peptides' were entered into the PubMed

search field (https://www.ncbi.nlm.nih.gov/pubmed/). Only linear peptides between 2 to 11 residues

containing natural amino acids were shortlisted and synthesized by Mimotopes (Australia) to >95%

purity.

2.2 MIC assay

The MICs of test compounds were determined using the broth microdilution protocol from the Clinical

and Laboratory Standards Institute (CLSI) guidelines [CLSI 2012]. Briefly, bacteria were grown fresh

from frozen stock on Mueller Hinton 2 (MH2) agar at 37°C. After overnight incubation, 5 colonies were

selected to grow in cation-adjusted MH2 broth in a shaking incubator (220 RPM) at 37°C. Cells were

grown to an optical density (OD600) of 0.15–0.16 determined using a spectrophotometer (Molecular

Devices Spectra Max Plus), which corresponds to ~ 1 x 108 CFU/mL. Test compounds were

constituted into 4 mM DMSO stock solutions and then subjected to 2-fold serial dilution in a 96-well

plate with concentrations ranging from 100 to 0.195 M in duplicates. 50 L of microbial culture

containing ~ 1 x 106 CFU/mL of microbes in the respective broths was introduced into each well

containing 50 L of compound solution. After an overnight incubation at 35°C, (140 RPM), OD600

measurements were conducted using the microplate spectrophotometer. MIC was defined as the

lowest antibiotic or peptide concentration (M) required to inhibit visible microbial growth.

2.3 Bactericidal/static determination assay

After the MICs of test compounds were determined using the CLSI microdilution protocol, the entire

well contents (100 L) corresponding to 4x MIC of peptide 50 (12.5 M) and retapamulin (0.78 M)

were transferred and spread on fresh sterile MH2 agar plates. The plates were incubated overnight at

37°C and the number of colony forming units (CFU) was determined the next day. A compound was

classified as bactericidal when ≥ 99.9% cell death was observed.

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2.4 Time-kill assay

Bacteria cells were grown to 1 x 108 CFU/mL in MH2 broth and then diluted 200-fold into 5 mL

aliquots to a density of 5 x 105 CFU/mL in a flask. Test compounds pre-dissolved in DMSO

corresponding to 4x their MICs [peptide 50 (12.5 M) and retapamulin (0.78 M)] were then added

into each flask with a final DMSO concentration of 2.5% v/v. The negative control was 2.5% DMSO.

The flasks were incubated in a shaking incubator (220 RPM) at 37°C. Samples (100 L) were taken

hourly from the flasks between t = 0 and t = 6 h. The samples were serially diluted in MH2 broth

before plating on MH2 agar and incubated overnight at 37°C. A bacteria count for each plate was

conducted the next day. A graph of Log CFU/mL was plotted against time to obtain the time-kill graph.

2.5 Human epidermal keratinocyte viability assay

Normal human epidermal keratinocytes (NHEK) were purchased from Lonza (catalog #00192627)

and cultured in keratinocyte growth medium (Lonza KGM-Gold BulletKit) containing growth

supplements (Lonza ReagentPack) at 5% CO2 (37°C). The cell viability assay was performed using

the CellTiter-Glo Luminescent Cell Viability Assay protocol from Promega [www.promega.com].

Briefly, NHEK were treated with test compounds in keratinocyte growth medium in 96-well plates

(Thermo Scientific Nunclon Delta). Treated cells were incubated for 5 days at 5% CO2 (37°C).

Promega CellTiter-Glo Reagent was added and plates were shaken on an orbital shaker for 2 hours.

Next, well contents (100 L) from each well were transferred into opaque 96-well plates (PerkinElmer

OptiPlate-96) and luminescence measured by a microplate reader (Tecan Safire2).

2.6 Spray formulations

The retapamulin spray formulation was made by dissolving retapamulin (21.17 mg) in ethanol

(1047.92 mg) and sterile water (1047.92 mg) followed by stirring at room temperature to obtain a

clear solution (1% w/w; 8.92 mg/mL) before transferring into a spray bottle.

The peptide spray formulation was made by dissolving peptide 50 (3.09 mg) in ethanol (152.96 mg)

and sterile water (152.96 mg) followed by stirring at room temperature to obtain a clear solution (1%

w/w; 8.92 mg/mL) before transferring into a spray bottle.

6

The blank spray formulation was made by mixing ethanol (1000 mg) and sterile water (1000 mg)

before transferring into a spray bottle.

2.7 Topical formulation MRSA growth inhibition assay

Five mupirocin-resistant MRSA (ATCC-BAA-1556) colonies were grown in cation-adjusted MH2 broth

and incubated in a shaking incubator (220 RPM) at 37 °C. Cells were grown to an OD600 of 0.60 - 0.90

before being spread on a plate containing sterile MH2 agar (15 mL) using a sterile disposable cotton

bud. Next, the formulations were sprayed onto the agar surface (145 ± 2 L) with a single quick

depression of the pump from a distance of 10 cm. The MH2 agar plates were incubated overnight at

37 °C and visually inspected the next day. Next, swabs were taken from the surfaces with sterile

disposable cotton buds and streaked on sterile MH2 agar surface. The plates were incubated

overnight at 37 °C followed by visual inspection for bacteria colonies the next day.

3. Results and Discussion

Our PubMed literature search over the last decade (2006 - 2015) uncovered 61 AMPs with reported

bioactivities against various bacterial species. These were commercially synthesized and subjected to

a head-to-head MIC comparison against mupirocin-resistant MRSA (ATCC-BAA-1556) using 8 FDA-

approved topical antibiotics for comparison (Table 1).

[Place Table 1 here]

The FDA-approved topical antibiotics exhibited MICs ranging from <0.2 to >100 M against

mupirocin-resistant MRSA (Table 1) with the two most potent antibiotics being retapamulin (MIC <0.2

M) and neomycin (MIC 0.78 M). Retapamulin (Altabax®) is a pleuromutilin class narrow-spectrum

antibiotic which targets the bacterial 50S ribosome, inhibiting protein synthesis and is indicated for

treating skin infections caused by S. aureus (Rittenhouse et al 2006). Neomycin is an aminoglycoside

broad-spectrum antibiotic which targets the bacterial 30S ribosome, inhibiting protein synthesis and is

sold as a combination antibacterial ointment together with bacitracin zinc and polymyxin B

7

(Neosporin®). The ointment is used as a mupirocin substitute in the UK for treating skin infections

caused by mupirocin-resistant MRSA (Worby et al 2013). Interestingly, the remaining 6 antibiotics

tested (bacitracin, clindamycin, erythromycin, mupirocin, polymyxin B and tetracycline) showed either

weak or no activities against mupirocin-resistant MRSA (MIC ≥50 M), suggesting that the MRSA

strain used in this study was multidrug-resistant.

Of the 61 peptides tested, 11 peptides (18%) displayed single-digit micromolar MICs against

mupirocin-resistant MRSA. It was notable that peptides with 8 or less residues exhibited poor MICs

(≥25 M) with the exception of the arginine and tryptophan-rich peptide 13 (RWRWRWRW-NH2)

which possessed moderate activity (MIC 12.5 M), close to the reported value of 5.1 M against

mupirocin-susceptible MRSA ST247 (Liu et al 2007). To achieve single-digit micromolar MICs against

MRSA, the MIC data in Table 1 suggested that AMPs needed to have at least 9 residues and contain

a minimum 3 basic residues (e.g., peptide 18; MIC 6.25 M). We also observed that peptides with C-

terminal carboxyl groups lacked bioactivity against mupirocin-resistant MRSA (peptides 36, 39, 41,

43, 44, 47 and 49; MICs >100 M). A plausible reason could be the anionic nature of bacterial

membranes due to the presence of anionic teichoic acids and phospholipids like phosphatidylglycerol,

resulting in electrostatic repulsion between the bacteria membrane surface and the AMP (Melo et al

2009; Yeaman and Yount 2003; Wimley 2010; Zhao et al 2008). For the same reason, uncharged

peptides 35 and 36 and the singly-charged cationic peptide 47 were also found to be impotent (MICs

>100 M), suggesting that the presence of basic residues in the peptide was important. Interestingly,

peptides containing 7 or more basic residues (peptides 34, 43-46, 58-61) were found to have either

no activity or possess very weak activity (MICs >50 M). We postulate that the high number of

cationic residues could have rendered these peptides too hydrophilic to penetrate the hydrophobic

lipid core of the bacteria membrane, preventing membrane insertion and subsequent membrane

disruption.

The two most potent peptides, 37 and 50, exhibited MICs of 3.13 M, 4-fold more potent than the 12-

residue clinical candidate Omiganan (Table 1). Although less potent than retapamulin (MIC <0.2 M)

and neomycin (MIC 0.78 M), peptides 37 and 50 were significantly more potent than the rest of the

8

FDA-approved topical antibacterial drugs tested (Table 1), suggesting that both AMPs could

potentially be developed into antibacterial agents.

Peptide 37, also known as Anoplin W5K8, is a 10-residue synthetic peptide derived from anoplin, a

peptide originally isolated from the venom of the Japanese Spider Wasp Anoplius samariensis (Munk

et al 2013; Konno et al 2001). Its MIC of 3.13 M is close to the literature's reported MIC of 2.5 M

against a mupirocin-susceptible MRSA strain (ATCC-33591) (Munk et al 2013). However, peptide 37

was also reported to be highly haemolytic towards human erythrocytes with an EC50 of 11.6 M (Munk

et al 2013), rendering it unsuitable for further drug development.

Peptide 50, also known as peptide 3.1, is a de novo-designed synthetic peptide based on Edmundson

helical wheel projection models (Kang et al 2009). This 11-residue peptide was reported to exhibit an

MIC of 1.6 g/mL (1.2 M) against methicillin-susceptible S. aureus (ATCC-6538p) (Kang et al 2009),

close to the 3.13 M MIC obtained in our experiments against mupirocin-resistant MRSA.

Interestingly, peptide 50 lacked the high hemolytic activity exhibited by peptide 37, causing only

14.9% hemolysis at 100 g/mL (75 M) (Kang et al 2009). Based on these results, peptide 50 was

selected for further MIC testing against a panel of clinical MRSA isolates (Table 2).

[Place Table 2 here]

The MRSA test panel (Table 2) revealed peptide 50 to possess MICs of 3.13 M against all 10 MRSA

strains, including mupirocin-resistant and -susceptible strains. Interestingly, the only FDA-approved

topical antibacterial drug with activity against all 10 MRSA strains was retapamulin, exhibiting

impressive sub-micromolar MICs (<0.2 M; Table 2). Surprisingly, neomycin was found to be inactive

(MIC ≥50 M) in 7 out of the 10 MRSA strains tested. A plausible reason could be that neomycin is an

old antibiotic discovered in 1949 (Waksman et al 1949) and its availability as an over-the-counter

topical drug could have resulted in the development of resistance in many MRSA strains. Based on

the data from Table 2, retapamulin was selected as the comparator for the rest of our experiments.

9

A bactericidal/static determination assay was next conducted on peptide 50 and retapamulin. A

bactericidal mechanism of action is preferable over a bacteriostatic one as this reduces the chance of

resistance development. At 4x MIC, peptide 50 was found to be bactericidal while retapamulin was

found to be bacteriostatic, in agreement with the literature (Rittenhouse et al 2006) (Fig. 1). We

postulate that peptide 50's membrane-disrupting mechanism of action was responsible for its

bactericidal effect. This suggested that peptide 50 was advantageous over retapamulin as an

antibacterial drug.

[Place Fig. 1 here]

Next, peptide 50 and retapamulin were subjected to a time-kill assay using mupirocin-resistant

MRSA. Results revealed peptide 50 to be rapidly bactericidal, achieving a 6-log CFU reduction in one

hour (Fig. 2). Retapamulin, in contrast, displayed a slow-killing mechanism of action, yielding a 1-log

CFU reduction in six hours. An ideal antibacterial drug should possess fast-killing mechanism of

action as this reduces the chance of resistance development and treatment duration. Based on Fig. 2,

our data suggests that peptide 50 was advantageous over retapamulin and can potentially be

developed as an antibacterial drug.

[Place Fig. 2 here]

There are concerns that AMPs with a membrane-disrupting mode of action may also lyse human

cells, precluding them from further development as therapeutics. A well-studied example is the 26-

residue lytic peptide, melittin, isolated from honey bee venom (Walsh et al 2011). As peptide 50 could

potentially be developed into a topical agent, this warranted an investigation into its degree of toxicity

towards normal human epidermal keratinocytes (Fig. 3). Melittin and retapamulin were included for

comparison. Propylene glycol, an FDA-approved topical formulation solvent found commonly in

topical gels, creams and lotions, was used as a negative control.

[Place Fig. 3 here]

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Fig.3 revealed melittin to be highly toxic towards human epidermal keratinocytes, killing 50% of the

cells at a concentration of 0.6 M. In contrast, peptide 50 was found to be ~80-fold less toxic, killing

50% of the cells at a concentration of 52 M. Interestingly, the FDA-approved topical antibacterial,

retapamulin, killed 50% of the cells at a concentration of 27 M, ~2-fold more toxic than peptide 50. A

plausible reason for retapamulin's toxicity towards human keratinocytes may be that being a bacterial

ribosome inhibitor, it may also inhibit eukaryotic ribosomes at high concentrations. Indeed, an

European Medicines Agency (EMA) toxicity report revealed retapamulin to be a skin irritant even

when applied below the proposed clinical concentration of 1% (w/w) on rabbit skin although skin

irritation was not observed on minipig skin (EMA 2007). Overall, the keratinocyte toxicity profile in Fig.

3 suggested that peptide 50 can potentially be developed as a topical agent.

Lastly, peptide 50 and retapamulin were formulated into spray formulations (1% w/w) and subjected

to an in vitro MRSA growth inhibition assay using agar plates to gauge peptide 50’s potential for

further development. In this experiment, ~145 L of the respective formulations were sprayed onto

MH2 agar plates pre-streaked with mupirocin-resistant MRSA. The plates were incubated for 24 h.

before observing the presence of bacterial growth (Fig. 4).

[Place Fig. 4 here]

Surprisingly, no bacteria growth inhibition was observed on the agar treated with the blank spray after

an overnight incubation (Fig. 4B), suggesting that a 50% ethanol solution lacked antibacterial

properties. In contrast, the spray formulations containing 1% w/w retapamulin and 1% w/w peptide 50

inhibited bacterial growth as illustrated in Figs. 4C and 4D respectively, suggesting both formulations

possessed antibacterial properties. To test for the presence of viable bacteria (invisible to the naked

eye) on the agar surface of both plates, swabs were taken from the agar surfaces, streaked on

separate sterile agar plates and incubated overnight. Bacteria growth was observed for the

retapamulin formulation (Fig. 4E), supporting our earlier findings that it was bacteriostatic (Fig. 1A). In

contrast, the streak plate of peptide 50 was devoid of bacteria (Fig. 4F), supporting our earlier findings

of its bactericidal mode of action (Fig. 1B). This also suggests that peptide 50 possess an advantage

over retapamulin as an antibacterial agent as viable bacteria can potentially develop resistance.

11

Conclusions

Based on the experimental results reported herein, we show that peptide 50 can potentially be

developed into a topical antibacterial drug for the treatment of mupirocin-resistant MRSA skin

infections. Its rapid bactericidal mode of action and short length gives it an edge over the FDA-

approved topical retapamulin and the clinical AMP candidate Omiganan respectively. Suggested

future work could involve structure-activity relationship studies including the use of unnatural amino

acids and the use of animal skin infection models.

Acknowledgements

This work was supported by the A*STAR Biomedical Research Council (Singapore).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Statements

The work described herein does not involve human participants or animals. Informed consent not

applicable.

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References

Abbassi F, Lequin O, Piesse C, Goasdoué N, Foulon T, Nicolas P et al (2010) Temporin-SHf, a new

type of phe-rich and hydrophobic ultrashort antimicrobial peptide. J Biol Chem 285:16880-16892.

Ahmad A, Ahmad E, Rabbani G, Haque S, Arshad M, Rizwan HK (2012) Identification and design of

antimicrobial peptides for therapeutic applications. Curr Protein Pept Sci 13:211-223.

Antonov NK, Garzon MC, Morel KD, Whittier S, Planet PJ, Lauren CT (2015) High prevalence of

mupirocin resistance in Staphylococcus aureus isolates from a pediatric population. Antimicrob

Agents Chemother 59:3350-3356.

Asoodeh A, Zardini HZ, Chamani J (2012) Identification and characterization of two novel

antimicrobial peptides, temporin-Ra and temporin-Rb, from skin secretions of the marsh frog Rana

ridibunda J Pept Sci 18:10-16.

Bai Y, Liu S, Jiang P, Zhou L, Li J, Tang C et al (2009) Structure-dependent charge density as a

determinant of antimicrobial activity of peptide analogues of defensin. Biochemistry 48:7229-7239.

Brogden NK, Brogden KA (2011) Will new generations of modified antimicrobial peptides improve

their potential as pharmaceuticals? Int J Antimicrob Agents 38:217-225.

Catiau L, Traisnel J, Delval-Dubois V, Chihib NE, Guillochon D, Nedjar-Arroume N (2011a) Minimal

antimicrobial peptidic sequence from hemoglobin alpha-chain: KYR. Peptides 32:633-638.

Catiau L, Traisnel J, Chihib NE, Le Flem G, Blanpain A, Melnyk O, Guillochon D, Nedjar-Arroume N

(2011b) RYH: a minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an

antimicrobial activity. Peptides 32:1463-1468.

Chan DI, Prenner EJ, Vogel HJ (2006) Tryptophan- and arginine-rich antimicrobial peptides:

structures and mechanisms of action. Biochim Biophys Acta 1758:1184-1202.

13

Chen C, Pan F, Zhang S, Hu J, Cao M, Wang J et al (2010) Antibacterial activities of short designer

peptides: a link between propensity for nanostructuring and capacity for membrane destabilization.

Biomacromolecules 11:402-411.

Cherkasov A, Hilpert K, Jenssen H, Fjell CD, Waldbrook M, Mullaly SC et al (2009) Use of artificial

intelligence in the design of small peptide antibiotics effective against a broad spectrum of highly

antibiotic-resistant superbugs. ACS Chem Biol 4:65-74.

Clinical and Laboratory Standards Institute (2012) Approved standard ninth edition.

Document M07-A9, Methods for dilution antimicrobial susceptibility tests for bacteria that grow

aerobically, vol. 32, CLSI, Wayne, PA. No. 2.

Coates T, Bax R, Coates A. Nasal decolonization of Staphylococcus aureus with mupirocin:

strengths, weaknesses and future prospects (2009) J Antimicrob Chemother 64:9-15.

De La Fuente-Núñez C, Korolik V, Bains M, Nguyen U, Breidenstein EB, Horsman S et al (2012)

Inhibition of bacterial biofilm formation and swarming motility by a small synthetic cationic peptide.

Antimicrob Agents Chemother 56:2696-2704.

Dryden MS (2010) Complicated skin and soft tissue infection, J Antimicrob Chemother 65 (Suppl. 3):

iii35-44.

Eltringham I (1997) Mupirocin resistance and methicillin-resistant Staphylococcus aureus (MRSA). J

Hosp Infect 35:1-8.

EMA: European public assessment report (EPAR) of Altargo (2007)

www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_Discussion/human/000757/

WC500024406.pdf Accessed 5 December 2016

14

Friden T (2013) Antibiotic resistance threats in the United States, 2013.

www.cdc.gov/drugresistance/threat-report-2013/ Accessed 5 December 2016

Godballe T, Mojsoska B, Nielsen HM, Jenssen H (2015) Antimicrobial activity of GN peptides and

their mode of action. Biopolymers 106:172-183.

Gopal R, Seo CH, Song PI, Park Y (2013) Effect of repetitive lysine-tryptophan motifs on the

bactericidal activity of antimicrobial peptides. Amino Acids 44:645-660.

Gould IM (2006) Costs of hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) and

its control. Int J Antimicrob Agents 28:379-384.

Guzmán F, Marshall S, Ojeda C, Albericio F, Carvajal-Rondanelli P (2013) Inhibitory effect of short

cationic homopeptides against gram-positive bacteria. J Pept Sci 19:792-800.

Hancock REW, Sahl H (2006) Antimicrobial and host-defense peptides as new antiinfective

therapeutic strategies. Nat Biotechnol 24:1551-1557.

Hetem DJ, Bonten MJM (2013) Clinical relevance of mupirocin resistance in Staphylococcus aureus.

J Hosp Infect 85:249-256.

Hilpert K, Volkmer-Engert R, Walter T, Hancock REW (2005) High-throughput generation of small

antibacterial peptides with improved activity. Nat Biotechnol 23:1008-1012.

Hilpert K, Elliott M, Jenssen H, Kindrachuk J, Fjell CD, Körner J et al (2009) Screening and

characterization of surface-tethered cationic peptides for antimicrobial activity. Chem Biol 16:58-69.

Hon PY, Koh TH, Tan TY, Krishnan P, Leong JW, Jureen R, Chan J, Tee NW, Murugesh J, Chan KS,

Hsu LY (2014) Changing molecular epidemiology and high rates of mupirocin resistance among

15

meticillin-resistant Staphylococcus aureus in Singaporean hospitals. J Glob Antimicrob Resist 2:53-

55.

Hou X, Du Q, Li R, Zhou M, Wang H, Wang L et al (2015) Feleucin-BO1: a novel antimicrobial non-

apeptide amide from the skin secretion of the toad, Bombina orientalis, and design of a potent broad-

spectrum synthetic analogue, feleucin-K3. Chem Biol Drug Des 85:259-267.

Iannucci NB, Curto LM, Albericio F, Cascone O, Delfino JM (2014) Structural glance into a novel anti-

staphylococcal peptide. Biopolymers 102:49-57.

Juba M, Porter D, Dean S, Gillmor S, Bishop B (2013) Characterization and performance of short

cationic antimicrobial peptide isomers. Biopolymers 100:387-401.

Kang SJ, Won HS, Choi WS, Lee BJ (2009) De novo generation of antimicrobial LK peptides with a

single tryptophan at the critical amphipathic interface. J Pept Sci 15:583-588.

Kinch MS, Patridge E, Plummer M, Hoyer D (2014) An analysis of FDA-approved drugs for infectious

disease: antibacterial agents. Drug Discov Today 19:1283-1287.

Konno K, Hisada M, Fontana R, Lorenzi CCB, Naoki H, Itagaki Y, Miwa A,

Kawai N, Nakata Y, Yasuhara T, Neto JR, De Azevedo WF Jr, Palma MS,

Nakajima T (2001) Anoplin, a novel antimicrobial peptide from the venom of

the solitary wasp Anoplius samariensis. Biochim Biophys Acta 1550:70-80.

Kukowska M, Kukowska-Kaszuba M, Dzierzbicka K (2015) In vitro studies of antimicrobial activity of

Gly-His-Lys conjugates as potential and promising candidates for therapeutics in skin and tissue

infections. Bioorg Med Chem Lett 25:542-546.

Lee SH, Kim SJ, Lee YS, Song MD, Kim IH, Won HS (2011) De novo generation of short

antimicrobial peptides with simple amino acid composition. Regul Pept 166:36-41.

16

Lepainteur M, Royer G, Bourrel AS, Romain O, Duport C, Doucet-Populaire F, Decousser JW (2013)

Prevalence of resistance to antiseptics and mupirocin among invasive coagulase-negative

staphylococci from very preterm neonates in NICU: the creeping threat? J Hosp Infect 83:333-336.

Li X, Li P, Saravanan R, Basu A, Mishra B, Lim SH et al (2014) Antimicrobial functionalization of

silicone surfaces with engineered short peptides having broad spectrum antimicrobial and salt-

resistant properties. Acta Biomater 10:258-266.

Lim K, Chua RR, Saravanan R, Basu A, Mishra B, Tambyah PA et al (2013) Immobilization studies of

an engineered arginine-tryptophan-rich peptide on a silicone surface with antimicrobial and antibiofilm

activity. ACS Appl Mater Interfaces 5:6412-22.

Liu Z, Brady A, Young A, Rasimick B, Chen K, Zhou C, Kallenbach NR (2007) Length effects in

antimicrobial peptides of the (RW)n series. Antimicrob Agents Chemother. 51:597-603.

Lung FD, Wang KS, Liao ZJ, Hsu SK, Song FY, Liou CC et al (2012) Discovery of potent

antimicrobial peptide analogs of Ixosin-B. Bioorg Med Chem Lett 22:4185-4188.

Marr AK, Gooderham WJ, Hancock REW (2006) Antibacterial peptides for therapeutic use: obstacles

and realistic outlook. Curr Opin Pharmacol 6:468-472.

Melo MN, Ferre R, Castanho MARB (2009) Antimicrobial peptides: linking

partition, activity and high membrane-bound concentrations, Nat Rev

Microbiol 7:245-250.

Mohamed MF, Hamed MI, Panitch A, Seleem MN (2014) Targeting methicillin-resistant

Staphylococcus aureus with short salt-resistant synthetic peptides. Antimicrob Agents Chemother

58:4113-4122.

17

Moran GJ, Krishnadasan A, Gorwitz RJ, Fosheim GE, McDougal LK, Carey RB, Talan DA (2006)

Methicillin-resistant S. aureus infections among patients in the emergency department. N Engl J Med

355:666-674.

Munk JK, Uggerhøj LE, Poulsen TJ, Frimodt-Møller N, Wimmer R, Nyberg NT, Hansen PR (2013)

Synthetic analogs of anoplin show improved antimicrobial activities. J Pept Sci 19:669-675.

Murugan RN, Jacob B, Kim EH, Ahn M, Sohn H, Seo JH et al (2013) Non hemolytic short

peptidomimetics as a new class of potent and broad-spectrum antimicrobial agents. Bioorg Med

Chem Lett 23:4633-4636.

Naidoo VB, Rautenbach M (2013) Self-assembling organo-peptide bolaphiles with KLK tripeptide

head groups display selective antibacterial activity. J Pept Sci 19:784-791.

Ong, ZY, Gao SJ, Yang YY (2013) Short synthetic β-sheet forming peptide amphiphiles as broad

spectrum antimicrobials with antibiofilm and endotoxin neutralizing capabilities. Adv Funct Mater

23:3682-3692.

Park KH, Park Y, Park IS, Hahm KS, Shin SY (2008) Bacterial selectivity and plausible mode of

antibacterial action of designed Pro-rich short model antimicrobial peptides. J Pept Sci 14:876-882.

Park KH, Nan YH, Park Y, Kim JI, Park IS, Hahm KS et al (2009) Cell specificity, anti-inflammatory

activity, and plausible bactericidal mechanism of designed Trp-rich model antimicrobial peptides.

Biochim Biophys Acta 1788:1193-1203.

Park SY, Kim SM, Park SD (2012) The prevalence, genotype and antimicrobial susceptibility of high-

and low-Level mupirocin resistant methicillin-resistant Staphylococcus aureus. Ann Dermatol 24:32-

38.

18

Pasupuleti M, Schmidtchen A, Chalupka A, Ringstad L, Malmsten M (2009) End-tagging of ultra-short

antimicrobial peptides by W/F stretches to facilitate bacterial killing. PLoS One 4:e5285.

Patel JB, Gorwitz RJ, Jernigan JA (2009) Mupirocin resistance. Clin Infect Dis 49:935-941.

Poovelikunnel T, Gethin G, Humphreys H (2015) Mupirocin resistance: clinical implications and

potential alternatives for the eradication of MRSA. J Antimicrob Chemother 70:2681-2692.

Qi X, Zhou C, Li P, Xu W, Cao Y, Ling H et al (2010) Novel short antibacterial and antifungal peptides

with low cytotoxicity: Efficacy and action mechanisms. Biochem Biophys Res Commun 398:594-600.

Ramón-García S, Mikut R, Ng C, Ruden S, Volkmer R, Reischl M et al (2013) Targeting

Mycobacterium tuberculosis and other microbial pathogens using improved synthetic antibacterial

peptides. Antimicrob Agents Chemother 57:2295-2303.

Rathinakumar R, Walkenhorst WF, Wimley WC (2009) Broad-spectrum antimicrobial peptides by

rational combinatorial design and high-throughput screening: the importance of interfacial activity. J

Am Chem Soc 131:7609-7617.

Rittenhouse S, Biswas S, Broskey J, McCloskey L, Moore T, Vasey S, West J, Zalacain M, Zonis R,

Payne D (2006) Selection of Retapamulin, a novel pleuromutilin for topical use. Antimicrob Agents

Chemother 50:3882-3885.

Romanelli A, Moggio L, Montella RC, Campiglia P, Iannaccone M, Capuano F et al (2011) Peptides

from royal jelly: studies on the antimicrobial activity of jelleins, jelleins analogs and synergy with

temporins. J Pept Sci 17:348-352.

Sader HS, Fedler KA, Rennie RP, Stevens S, Jones RN (2004) Omiganan pentahydrochloride (MBI

226), a topical 12-amino-acid cationic peptide: spectrum of antimicrobial activity and measurements

of bactericidal activity. Antimicrob Agents Chemother 48:3112-3118.

19

Saravanan R, Li X, Lim K, Mohanram H, Peng L, Mishra B et al (2014) Design of short membrane

selective antimicrobial peptides containing tryptophan and arginine residues for improved activity,

salt-resistance and biocompatibility. Biotechnol Bioeng 111:37-49.

Shin S, Kim JK, Lee JY, Jung KW, Hwang JS, Lee J et al (2009) Design of potent 9-mer antimicrobial

peptide analogs of protaetiamycine and investigation of mechanism of antimicrobial action. J Pept Sci

15:559-568.

Singh K, Kumar S, Shekhar S, Dhawan B, Dey S (2014) Synthesis and biological evaluation of novel

peptide BF2 as an antibacterial agent against clinical isolates of vancomycin-resistant enterococci. J

Med Chem 57:8880-8885.

Spindler EC, Hale JD, Giddings TH Jr, Hancock REW, Gill RT (2011) Deciphering the mode of action

of the synthetic antimicrobial peptide Bac8c. Antimicrob Agents Chemother 55:1706-1716.

Stevens DL, Bisno AL, Chambers HF, Everett ED, Dellinger P, Goldstein EJ, Gorbach SL,

Hirschmann JV, Kaplan EL, Montoya JG, Wade JC (2005) Practice guidelines for the diagnosis and

management of skin and soft-tissue infections. Clin Infect Dis 41:1373-1406.

Sundriyal S, Sharma RK, Jain R, Bharatam PV (2008) Minimum requirements of hydrophobic and

hydrophilic features in cationic peptide antibiotics (CPAs). J Mol Model 14:265-278.

Torcato IM, Huang YH, Franquelim HG, Gaspar D, Craik DJ, Castanho MA et al (2013) Design and

characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-

negative and Gram-positive bacteria. Biochim Biophys Acta 1828:944-955.

Upton A, Lang S, Heffernan H (2003) Mupirocin and Staphylococcus aureus: a recent paradigm of

emerging antibiotic resistance. J Antimicrob Chemother 51:613-617.

20

Waksman, SA, Lechevalier, HA, Harris, DA (1949) Neomycin - production and antibiotic properties. J

Clin Invest 28:934-939.

Walsh EG, Maher S, Devocelle M, O'Brien PJ, Baird AW, Brayden DJ (2011) High content analysis to

determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs. Peptides

32:1764-1773.

Wang Y, Chen J, Zheng X, Yang X, Ma P, Cai Y et al (2014) Design of novel analogues of short

antimicrobial peptide anoplin with improved antimicrobial activity. J Pept Sci 20:945-951.

Wei SY, Wu JM, Kuo YY, Chen HL, Yip BS, Tzeng SR et al (2006) Solution structure of a novel

tryptophan-rich peptide with bidirectional antimicrobial activity. J Bacteriol 188:328-334.

Wimley WC (2010) Describing the mechanism of antimicrobial peptide action with

the interfacial activity model. ACS Chem Biol 5:905-917.

Worby CJ, Jeyaratnam D, Robotham JV, Kypraios T, O'Neill PD, De Angelis D, French G, Cooper BS

(2013) Estimating the effectiveness of isolation and decolonization measures in reducing transmission

of methicillin-resistant Staphylococcus aureus in hospital general wards. Am J Epidemiol 177:1306-

1313.

Yang X, Wang Y, Lee WH, Zhang Y (2013) Antimicrobial peptides from the venom gland of the social

wasp Vespa tropica. Toxicon 74:151-157.

Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and

resistance. Pharmacol Rev 55:27-55.

Yeung ATY, Gellatly SL, Hancock REW (2011) Multifunctional cationic host defence peptides and

their clinical applications. Cell Mol Life Sci 68:2161-2176.

21

Youn SH, Lee SS, Kim S, Lee JA, Kim BJ, Kim J, Han HK, Kim JS (2015) Drug utilization review of

mupirocin ointment in a Korean university-affiliated hospital. Korean J Intern Med 30:515-520.

Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389-395.

Zhang L, Falla TJ (2006) Antimicrobial peptides: therapeutic potential. Expert Opin Pharmacother

7:653-663.

Zhao W, Róg T, Gurtovenko AA, Vattulainen I, Karttunen M (2008) Role of phosphatidylglycerols in

the stability of bacterial membranes. Biochimie 90:930-938.

Zhou L, Liu SP, Chen LY, Li J, Ong LB, Guo L et al (2011) The structural parameters for antimicrobial

activity, human epithelial cell cytotoxicity and killing mechanism of synthetic monomer and dimer

analogues derived from hBD3 C-terminal region. Amino Acids 40:123-133.

Zorko M, Japelj B, Hafner-Bratkovic I, Jerala R (2009) Expression, purification and structural studies

of a short antimicrobial peptide. Biochim Biophys Acta 1788:314-323.

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Captions

Table1 MICs (M) of FDA-approved topical antibacterial drugs and peptides against mupirocin-

resistant MRSA (ATCC-BAA-1556)

Table2 MICs (M) of FDA-approved antibacterial topical agents and peptide 50 against various

clinical strains of MRSA. USA100 (ATCC-BAA-1681); USA200 (ATCC-BAA-1750), USA300 (ATCC-

BAA-1756); USA400 (ATCC-BAA-1707); EMRSA-15 (ATCC-BAA-2762); ST247 (ATCC-BAA-44);

VISA (ATCC-700699)

Fig.1 Bactericidal/static determination assay at 4x MIC: (A) retapamulin; (B) peptide 50 on mupirocin-

resistant MRSA (ATCC-BAA-1556)

Fig.2 Time-kill assay using mupirocin-resistant MRSA (ATCC-BAA-1556)

Fig.3 Normal human epidermal keratinocyte viability assay using peptides, retapamulin and

propylene glycol.

Fig.4 MRSA (ATCC-BAA-1556) agar plate growth inhibition assay: (A) spray bottle; (B) blank spray

formulation; (C) 1% w/w retapamulin spray formulation; (D) 1% w/w peptide 50 spray formulation; (E)

surface streak plate of plate C; (F) surface streak plate of plate D. Note that the dark rectangular

regions observed in panels C to F are the reflections of the camera due to the glossy agar surface

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