Potent Broad-Spectrum Antibacterial Activity of Amphiphilic Peptides against Multidrug-Resistant Bacteria

The emergence and prevalence of multidrug-resistant (MDR) bacteria particularly Gram-negative bacteria presents a global crisis for human health. Colistin and tigecycline were recognized as the last resort of defenses against MDR Gram-negative pathogens. However, the emergence and prevalence of MCR or Tet(X)-mediated acquired drug resistance drastically impaired their clinical efficacy. It has been suggested that antimicrobial peptides might act a crucial role in combating antibiotic resistant bacteria owing to their multiple modes of action and characteristics that are not prone to developing drug resistance. Herein, we report a safe and stable tryptophan-rich amphiphilic peptide termed WRK-12 with broad-spectrum antibacterial activity against various MDR bacteria, including MRSA, colistin and tigecycline-resistant Escherichia coli. Mechanistical studies showed that WRK-12 killed resistant E. coli through permeabilizing the bacterial membrane, dissipating membrane potential and triggering the production of reactive oxygen species (ROS). Meanwhile, WRK-12 significantly inhibited the formation of an E. coli biofilm in a dose-dependent manner. These findings revealed that amphiphilic peptide WRK-12 is a promising drug candidate in the fight against MDR bacteria.


Introduction
Pathogenic bacteria particularly Gram-negative pathogens such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii have been a major cause of systemic infections in clinics [1,2]. More alarmingly, these pathogens have developed multiple acquired resistance against current antimicrobial treatments [3][4][5]. For example, the acquisition of metallo-β-lactamases (MBLs) such as NDM-1 resulted in the emergence of carbapenem-resistant Gram-negative pathogens [6]. Mobile colistin resistance gene mcr-1 and its variants [7,8] protect Enterobacteriaceae from colistin killing, an important cationic antibiotic that is recognized as one of last-resort options against multidrug-resistant (MDR) Gram-negative bacteria [9]. In addition, recent study revealed that tet(X3/X4) genes located on plasmid confer high level tigecycline resistance in

Antibacterial Activity Tests
Minimum inhibitory concentrations (MICs) of peptides were determined by broth micro-dilution according to the CLSI2018 guideline [23]. Briefly, overnight strains were diluted 1:100 into Mueller-Hinton broth (MHB) and incubated at 37 • C with sharking at 200 rpm for 4 h. Subsequently, varying concentrations of drugs were mixed with an equal volume of bacterial suspensions in MHB containing approximately 1.5 × 10 6 colony-forming units per mL in a sterilized 96-well microtitre plate (Corning). After 18 h of incubation at 37 • C, MIC values were determined as the lowest concentrations of drugs with no visible growth of bacteria. For the minimum bactericidal concentration (MBC) assay, 50 µL of mixture was taken out from clear well in MIC assays, resuspended in fresh media and plated onto Mueller-Hinton agar (MHA) overnight at 37 • C. The MBC is defined as the lowest concentration of drugs that killed 99.9% of the bacterial cells. Experiments were performed with two biological replicates.
Salts and serum stability. For assessing the effect of salts and serum on the activity of WRK-12, three salts (including Na + , K + , Ca 2+ , final concentration 10 mM), 10% fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) were added into MHB for a following MIC assay.
Thermal, pH and proteolytic stability. WRK-12 was preincubated at different temperatures (from 25 to 121 • C), pH (from 2 to 12) or proteases (pepsin, trypsin and papain, final concentration 10 mg/mL) for 1 h. Samples after pH treatment were readjusted to pH = 7.2 to determine the residual antibacterial activity by MIC tests. Samples after proteases treatment were heated at 80 • C for 30 min, and centrifuged at 13,000× g for 30 min to precipitate proteases, and the residual antibacterial activity of supernatants was tested by MIC analysis.

Hemolysis Analysis
Hemolytic activity of all peptides was evaluated based on previous studies [24,25]. Sheep red blood cells (RBCs) were washed with 0.9% saline two times, re-suspended to obtain 8% red blood cell suspension and then mixed with increasing concentrations of AMPs at 37 • C for 1 h. Sterilized PBS and double-distilled water (ddH 2 O) were used as blank and positive control, respectively. The absorption of released hemoglobin was measured at 576 nm by an Infinite M200 Microplate reader (Tecan, Männedorf, Switzerland). Hemolysis rate was calculated by comparing the absorbance of the sample and the positive control after subtracting the blank control.

CD Measurements
Circular dichroism (CD) spectra of the peptides were recorded with a J-810 spectropolarimeter (Jasco, Tokyo, Japan) at 25 • C. The spectra were measured in 0.01 M PBS, 50 µM LPS or 50 mM SDS (final concentration, 100 µg/mL). The CD spectra were recorded at a wavelength of 190 to 260 nm, and the data are expressed as mean residue ellipticity 2.6. Outer Membrane Permeabilization E. coli 1F28 were grown overnight at 37 • C with shaking at 200 rpm. Bacterial cells were washed and resuspended with 5 mM HEPES to an OD 600 of 0.5 and incubated with fluorescent probe 1-N-phenylnaphthylamine (NPN, 0.1 µM) at 37 • C in a humidified atmosphere for 30 min [26]. Subsequently, 190 µL of probe-labelled cells were mixed with 10 µL WRK-12 (0 to 128 µg/mL) or colistin as a positive control (128 µg/mL) in a sterile 96-well black plate. After incubation at 37 • C in a humidified atmosphere for 60 min, fluorescence intensity was measured on an Infinite M200 Microplate reader (Tecan, Männedorf, Switzerland) (λexcitation = 350 nm, λemission = 420 nm).

Prevention of Biofilm Formation
The prevention of biofilm formation was assessed as described previously [28]. Briefly, bacteria (1 × 10 5 CFUs per mL) were exposed to WRK-12 solutions (with final concentrations ranging from 0.25 to 32 µg/mL). As an untreated control, bacteria were exposed to MHB without drugs. After 24 h incubation at 37 • C in a humidified atmosphere, planktonic bacteria were removed by phosphate buffer solution (PBS). Biofilms were fixed with methanol for 15 min, then sucked out the fixative and air dry naturally. Then, biofilms were stained with 0.1% crystal violet for 15 min, washed and dried naturally. Finally, 33% acetic acid was used to dissolve crystal violet. The optical density at 570 nm was determined as a measure of biofilm mass.

Characterizations of Engineered Peptides
A collection of tryptophan-rich linear peptides on the basis of MP196 (RWRWRW-NH 2 ) was designed by increasing peptide length or positive charge or hydrophobicity, replacing amino acids and/or N-terminal acetylation (Table 1). These peptides were synthesized via solid-phase peptide synthesis (SPPS), purified by reverse-phase high-performance liquid chromatography (RP-HPLC) ( Figure S1) and validated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) ( Figure S2). Chemical information and characterization of these engineered peptides are shown in Table 1. All peptides were accurately obtained with the purifies higher than 95%, and the molecular weights calculated by mass spectrometry are consistent with their theoretical molecular mass value, suggesting that all the engineered peptides were successfully synthesized. All engineered peptides belong to cationic AMPs, with 3 or 6 net charges. The percentage of acetonitrile at RP-HPLC elution was regarded as a relative measure of peptides' hydrophobicity. Accordingly, the majority of engineered peptides, except WK-6, exhibited higher hydrophobicity than MP196. The hydrophobicity order was listed as follows: WRK-12 > WL-9 > WR-6 > WV-9 > KW-6 > WKK-12 > WR-12 > MP196 > WK-6.
It has been suggested that cationicity and acetylation at N-terminal contribute to improve the activity of AMPs [29]; Therefore, three positive amino acids and/or acetylation at N-terminal were further introduced on the basis of two nonapeptides. As a consequence, we constructed three novel dodecapeptides including WR-12, WRK-12 and WKK-12 with acetylation at the N-terminus and amidation at the C-terminus. As expected, insertion of three positive amino acids in WL-9 (produce WR-12 or WRK-12) significantly increased its antibacterial activity with MICs decreasing by 4-fold change.
The wheel diagram showed these four active AMPs including WL-9, WR-12, WRK-12 and WKK-12 ( Figure 1A), which exhibited imperfect amphiphilic structures that possessed interrupted hydrophobic and cationic faces ( Figure 1B). Interestingly, although WRK-12 displayed high hydrophobicity, the solubility test showed that only WRK-12 has the highest solubility in 0.01 M PBS or water, whereas the other three peptides are slightly soluble in water. The introduction of Threonine (Thr, T) in WRK-12, an uncharged polar amino acid, may account for this paradox. Prior studies have illustrated that N-terminus acetylation has a critical effect on both peptide secondary structure and penetration ability on the bacterial membrane, as well as its in vivo efficacy [30,31]. Considering these points, WRK-12 and WKK-12 were chosen as potential candidates for our next study. The potentially charged residues, hydrophobicity residues and uncharged residues were marked as The potentially charged residues, hydrophobicity residues and uncharged residues were marked as blue, yellow and pink, respectively. The longer the arrow length, the greater the relative hydrophobic moments in the figure. MIC tests showed that WRK-12 and WKK-12 displayed antibacterial activity against MDR E. coli B2 with MIC values of 2 and 64 µg/mL, respectively. Subsequently, we investigated the antibacterial spectrum of WRK-12 and WKK-12 in a panel of MDR bacterial isolates. As shown in Table 2, we found that WRK-12 showed the broad-spectrum antibacterial activity against all test strains with MIC values from 2 to 4 µg/mL and MBC values from 2 to 8 µg/mL. These strains include hard-to-treat Gram-positive bacteria such as MRSA and VRE, MDR Gram-negative bacteria that are resistant to the last resort of clinically available antibiotics such as carbapenems, colistin and tigecycline. Specifically, for both mcr-1 and bla NDM-5 carrying colistin and carbapenems-resistant E. coli B2 (MIC of colistin, 8 µg/mL) and tet(X4)-positive tigecycline E. coli B3-1 (MIC of tigecycline, 32 µg/mL), WRK-12 also displayed potent activity, suggesting that the activity of WRK-12 is independent of these two resistance mechanisms. Considering the urgent need of novel antimicrobial agents for recent reported tigecycline-resistant Gram-negative bacteria in clinic, we next focused our insight on these strains. We investigated the activity of WRK-12 and WKK-12 against 11 tigecycline-resistant clinical isolates from a swine farm in 2019 (Table 3). Excitingly, WRK-12 displayed great antibacterial activity for all test strains, including important foodborne pathogen Shigella.  The secondary structure of the engineered peptides was tested by CD spectroscopy. As shown in Figure 2, WRK-12 exhibited a complete beta sheet conformation in phosphate buffer solution (PBS). LPS is one of important components in the Gram-negative bacterial outer membrane [32], and sodium dodecyl sulfate (SDS) micelle solution was used that mimicked negatively charged prokaryotic membrane-comparable environments. In the presence of 50 µM LPS and 50 mM SDS, the proportion of random and α-helical WRK-12 increased. This result indicated that the secondary structure of WRK-12 would present in hybrid form as it interacts with the bacterial membrane. LPS is one of important components in the Gram-negative bacterial outer membrane [32], and sodium dodecyl sulfate (SDS) micelle solution was used that mimicked negatively charged prokaryotic membrane-comparable environments. In the presence of 50 μM LPS and 50 mM SDS, the proportion of random and α-helical WRK-12 increased. This result indicated that the secondary structure of WRK-12 would present in hybrid form as it interacts with the bacterial membrane.

3.3.A Desirable Safety and Stability of WRK-12 against Bacteria
The hemolytic activity and instability of peptides are important challenges that hinder the clinical application of drugs [33]. In our study, hemolytic analysis showed that WRK-12 had the dispensable and lowest hemolytic activity (<5% at 128 μg/mL) on mammalian RBCs compared with the other three active AMPs (Figure 3), indicating higher selectivity of WRK-12 for bacteria other than mammalian cells. Although WR-12 showed comparable antibacterial activity with WRK-12, it displayed more than 50% of the hemolytic rate at 128 μg/mL. Great stability of antimicrobial peptides is a critical prerequisite for its in vivo efficacy. Thus, we evaluated the antibacterial activity of WRK-12 and WKK-12 against tigecycline resistant E. coli 1F28 and 1A34 in the presence of salt ions, serum and Dulbecco's Modified Eagle's Medium (DMEM) ( Table 4). There was not any loss of activity in the presence of monovalent cations (Na + and K + ), whereas divalent cation Ca 2+ , 10% serum or DMEM mildly reduced the activity of WRK-12 with the MICs increasing by 2 to 4-fold. The weakened activity by the divalent cation indicated that the action of WRK-12 may be correlated with membrane damage, because the outer membrane of Gramnegative bacteria could be stabilized with divalent cations particularly Ca 2+ and Mg 2+ [34]. By contrast,

A Desirable Safety and Stability of WRK-12 against Bacteria
The hemolytic activity and instability of peptides are important challenges that hinder the clinical application of drugs [33]. In our study, hemolytic analysis showed that WRK-12 had the dispensable and lowest hemolytic activity (<5% at 128 µg/mL) on mammalian RBCs compared with the other three active AMPs (Figure 3), indicating higher selectivity of WRK-12 for bacteria other than mammalian cells. Although WR-12 showed comparable antibacterial activity with WRK-12, it displayed more than 50% of the hemolytic rate at 128 µg/mL. LPS is one of important components in the Gram-negative bacterial outer membrane [32], and sodium dodecyl sulfate (SDS) micelle solution was used that mimicked negatively charged prokaryotic membrane-comparable environments. In the presence of 50 μM LPS and 50 mM SDS, the proportion of random and α-helical WRK-12 increased. This result indicated that the secondary structure of WRK-12 would present in hybrid form as it interacts with the bacterial membrane.

3.3.A Desirable Safety and Stability of WRK-12 against Bacteria
The hemolytic activity and instability of peptides are important challenges that hinder the clinical application of drugs [33]. In our study, hemolytic analysis showed that WRK-12 had the dispensable and lowest hemolytic activity (<5% at 128 μg/mL) on mammalian RBCs compared with the other three active AMPs (Figure 3), indicating higher selectivity of WRK-12 for bacteria other than mammalian cells. Although WR-12 showed comparable antibacterial activity with WRK-12, it displayed more than 50% of the hemolytic rate at 128 μg/mL. Great stability of antimicrobial peptides is a critical prerequisite for its in vivo efficacy. Thus, we evaluated the antibacterial activity of WRK-12 and WKK-12 against tigecycline resistant E. coli 1F28 and 1A34 in the presence of salt ions, serum and Dulbecco's Modified Eagle's Medium (DMEM) ( Table 4). There was not any loss of activity in the presence of monovalent cations (Na + and K + ), whereas divalent cation Ca 2+ , 10% serum or DMEM mildly reduced the activity of WRK-12 with the MICs increasing by 2 to 4-fold. The weakened activity by the divalent cation indicated that the action of WRK-12 may be correlated with membrane damage, because the outer membrane of Gramnegative bacteria could be stabilized with divalent cations particularly Ca 2+ and Mg 2+ [34]. By contrast, Great stability of antimicrobial peptides is a critical prerequisite for its in vivo efficacy. Thus, we evaluated the antibacterial activity of WRK-12 and WKK-12 against tigecycline resistant E. coli 1F28 and 1A34 in the presence of salt ions, serum and Dulbecco's Modified Eagle's Medium (DMEM) ( Table 4). There was not any loss of activity in the presence of monovalent cations (Na + and K + ), whereas divalent cation Ca 2+ , 10% serum or DMEM mildly reduced the activity of WRK-12 with the MICs increasing by 2 to 4-fold. The weakened activity by the divalent cation indicated that the action of WRK-12 may be correlated with membrane damage, because the outer membrane of Gram-negative bacteria could be stabilized with divalent cations particularly Ca 2+ and Mg 2+ [34]. By contrast, Na + , Ca 2+ , 10% serum or DMEM sharply impaired the weak antibacterial effect of WKK-12 against two tested isolates. In addition, medium containing 10% serum and DMEM was used to simulate an in vivo matrix environment. Only 2 to 4-fold increase of MIC values were found in these conditions. Next, we also assessed the thermal, pH and proteolytic stability of WRK-12. Surprisingly, WRK-12 completely retained its activity after treatment under 100 • C or pH (2 to 10) for 1 h ( Figure 4A,B), indicating that WRK-12 possesses great thermal and pH resistance. In contrast, a 50% activity reduction after exposure to 121 • C or alkaline environment (pH = 12) was observed. In the proteolytic stability study, we found that WRK-12 was resistant to pepsin treatment, but sensitive to trypsin and papain ( Figure 4C). High percentage of cationic amino acids in WRK-12 may account for this result. Together, these data suggested the great salt ions, serum, thermal and pH stability of WRK-12.
Na + , Ca 2+ , 10% serum or DMEM sharply impaired the weak antibacterial effect of WKK-12 against two tested isolates. In addition, medium containing 10% serum and DMEM was used to simulate an in vivo matrix environment. Only 2 to 4-fold increase of MIC values were found in these conditions. Next, we also assessed the thermal, pH and proteolytic stability of WRK-12. Surprisingly, WRK-12 completely retained its activity after treatment under 100 °C or pH (2 to 10) for 1 h ( Figure 4A,B), indicating that WRK-12 possesses great thermal and pH resistance. In contrast, a 50% activity reduction after exposure to 121 °C or alkaline environment (pH = 12) was observed. In the proteolytic stability study, we found that WRK-12 was resistant to pepsin treatment, but sensitive to trypsin and papain ( Figure  4C). High percentage of cationic amino acids in WRK-12 may account for this result. Together, these data suggested the great salt ions, serum, thermal and pH stability of WRK-12.

WRK-12 Targets LPS and Bacteria-Specific Phospholipids
Having shown the activity and stability of WRK-12, we set out to elucidate its bactericidal targets. Previous studies have demonstrated that cationic AMPs such as MSI-78 and LL-37 can disrupt the bacterial lipid bilayer structure through their electrostatic interactions with the polar headgroups [35,36]. Considering that WRK-12 is a cationic antibacterial peptide, we hypothesize that WRK-12 may damage the bacterial membrane through targeting the specific components of the

WRK-12 Targets LPS and Bacteria-Specific Phospholipids
Having shown the activity and stability of WRK-12, we set out to elucidate its bactericidal targets. Previous studies have demonstrated that cationic AMPs such as MSI-78 and LL-37 can disrupt the bacterial lipid bilayer structure through their electrostatic interactions with the polar headgroups [35,36]. Considering that WRK-12 is a cationic antibacterial peptide, we hypothesize that WRK-12 may damage the bacterial membrane through targeting the specific components of the bacterial membrane. To test this, we determined the effect of exogenous LPS or phospholipid supplements on the antibacterial activity of WRK-12 against E. coli 1F28. Consequently, we found that addition of LPS weakened WRK-12 activity in a dose-dependent manner ( Figure 5A), suggesting that LPS is a potential target of WRK-12. In addition to LPS, phospholipids including PE, phosphatidylglycerol (PG) and cardiolipin (CL) are important components of the bacterial plasma membrane, whereas phosphatidylcholine (PC) only presents in mammalian cell membranes [37]. Thus, we next performed a phospholipid competitive inhibition assay to assess the effect of exogenous lipids on WRK-12 activity. As a result, PG and CL drastically increased the MIC values of WRK-12 (16-fold at 16 µg/mL PG or CL), PE mildly impaired WRK-12 activity and no MIC changes were observed for CL ( Figure 5B). These results implied that WRK-12 exerted the activity through binding to LPS in the outer membrane, as well as PG and CL that were located in the bacterial cytoplasmic membrane. Meanwhile, the notion that PG and CL have a lower proportion in mammalian cell membranes give an explanation on its high selectivity on bacteria. Nevertheless, direct binding affinity assays between WRK-12 and these potential targets are still required to further strengthen these findings.
bacterial membrane. To test this, we determined the effect of exogenous LPS or phospholipid supplements on the antibacterial activity of WRK-12 against E. coli 1F28. Consequently, we found that addition of LPS weakened WRK-12 activity in a dose-dependent manner ( Figure 5A), suggesting that LPS is a potential target of WRK-12. In addition to LPS, phospholipids including PE, phosphatidylglycerol (PG) and cardiolipin (CL) are important components of the bacterial plasma membrane, whereas phosphatidylcholine (PC) only presents in mammalian cell membranes [37]. Thus, we next performed a phospholipid competitive inhibition assay to assess the effect of exogenous lipids on WRK-12 activity. As a result, PG and CL drastically increased the MIC values of WRK-12 (16-fold at 16 μg/mL PG or CL), PE mildly impaired WRK-12 activity and no MIC changes were observed for CL ( Figure 5B). These results implied that WRK-12 exerted the activity through binding to LPS in the outer membrane, as well as PG and CL that were located in the bacterial cytoplasmic membrane. Meanwhile, the notion that PG and CL have a lower proportion in mammalian cell membranes give an explanation on its high selectivity on bacteria. Nevertheless, direct binding affinity assays between WRK-12 and these potential targets are still required to further strengthen these findings.

WRK-12 Increases Membrane Permeability, Dissipates Membrane Potential and Induces ROS Production
To further elucidate membrane damage caused by WRK-12, we used a fluorescence probe 1-Nphenylnaphthylamine (NPN) to assess the effect of WRK-12 on the outer membrane permeability of E. coli. As shown in Figure 6A, WRK-12 at 128 μg/mL caused a significant fluorescence release, which was five-fold higher than colistin (128 μg/mL), indicating that WRK-12 strongly disrupted outer membrane permeability than colistin. Then, nucleic acid fluorescent dye propidium iodide (PI) was employed to evaluate the whole membrane permeability. As a result, WRK-12 led to a dosedependent increase of PI fluorescence, implying a remarkable damage to the bacterial membrane ( Figure 6B). By contrast, colistin (128 μg/mL) showed a week fluorescence increase, similar to 4 μg/mL WRK-12. Consistently, membrane disruption has been suggested as one of crucial mechanisms of action for AMPs killing [38]. Besides, WRK-12 significantly dissipated membrane potential (ΔΨ) ( Figure 6C), which was critical component of bacterial proton motive force [39]. ROS mediated killing has been evidenced to be important for bactericidal antibiotics [40]. Consistently, WRK-12 triggered the production of ROS in a concentration-dependent manner ( Figure 6D). In agreement with this observation, ROS scavenger NAC abolished the antibacterial activity of WRK-12 with MICs increased

WRK-12 Increases Membrane Permeability, Dissipates Membrane Potential and Induces ROS Production
To further elucidate membrane damage caused by WRK-12, we used a fluorescence probe 1-N-phenylnaphthylamine (NPN) to assess the effect of WRK-12 on the outer membrane permeability of E. coli. As shown in Figure 6A, WRK-12 at 128 µg/mL caused a significant fluorescence release, which was five-fold higher than colistin (128 µg/mL), indicating that WRK-12 strongly disrupted outer membrane permeability than colistin. Then, nucleic acid fluorescent dye propidium iodide (PI) was employed to evaluate the whole membrane permeability. As a result, WRK-12 led to a dose-dependent increase of PI fluorescence, implying a remarkable damage to the bacterial membrane ( Figure 6B). By contrast, colistin (128 µg/mL) showed a week fluorescence increase, similar to 4 µg/mL WRK-12. Consistently, membrane disruption has been suggested as one of crucial mechanisms of action for AMPs killing [38]. Besides, WRK-12 significantly dissipated membrane potential (∆Ψ) (Figure 6C), which was critical component of bacterial proton motive force [39]. ROS mediated killing has been evidenced to be important for bactericidal antibiotics [40]. Consistently, WRK-12 triggered the production of ROS in a concentration-dependent manner ( Figure 6D). In agreement with this observation, ROS scavenger NAC abolished the antibacterial activity of WRK-12 with MICs increased by 6-fold at 2.5 mM (Figure 7), suggesting that production of ROS is crucial for WRK-12 activity against MDR pathogens.

WRK-12 Inhibits Biofilm Formation
Biofilms produced by bacteria play a critical role in its pathogenicity and the development of drug resistance and have been implicated in chronic infections. Recent study has highlighted the possible use of AMPs to prevent biofilm formation or to treat established biofilms [28]. Intriguingly, WRK-12 dose-dependently inhibited the formation of biofilms by tigecycline-resistant E. coli 1F28

WRK-12 Inhibits Biofilm Formation
Biofilms produced by bacteria play a critical role in its pathogenicity and the development of drug resistance and have been implicated in chronic infections. Recent study has highlighted the possible use of AMPs to prevent biofilm formation or to treat established biofilms [28]. Intriguingly, WRK-12 dose-dependently inhibited the formation of biofilms by tigecycline-resistant E. coli 1F28 (Figure 8). Notably, a significant inhibition effect of WRK-12 on biofilm formation could be observed even though at a low concentration of drug (0.5 µg/mL). The biofilm mass, as measured using crystal violet staining after 24 h, drastically reduced after exposure to 2 µg/mL or higher concentrations of WRK-12. These results indicated that WRK-12 has a beneficial effect on the inhibition of biofilm formation. Figure 7. ROS scavenger NAC abolishes the antibacterial activity of WRK-12. MIC values of WRK-12 against E. coli 1F28 in the presence of increasing concentrations of NAC (N-acetylcysteine) were determined. Data were presented as mean ± SD from three independent experiments.

WRK-12 Inhibits Biofilm Formation
Biofilms produced by bacteria play a critical role in its pathogenicity and the development of drug resistance and have been implicated in chronic infections. Recent study has highlighted the possible use of AMPs to prevent biofilm formation or to treat established biofilms [28]. Intriguingly, WRK-12 dose-dependently inhibited the formation of biofilms by tigecycline-resistant E. coli 1F28 (Figure 8). Notably, a significant inhibition effect of WRK-12 on biofilm formation could be observed even though at a low concentration of drug (0.5 μg/mL). The biofilm mass, as measured using crystal violet staining after 24 h, drastically reduced after exposure to 2 μg/mL or higher concentrations of WRK-12. These results indicated that WRK-12 has a beneficial effect on the inhibition of biofilm formation. Figure 8. WRK-12 inhibits the formation of E. coli 1F28 biofilm in a concentration-dependent manner. Data were shown as mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001, determined by non-parametric one-way ANOVA. Data were shown as mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001, determined by non-parametric one-way ANOVA.

Conclusions
The emergence and prevalence of MDR pathogens call for novel and effective antimicrobial agents. In this study, we designed a series of tryptophan-rich amphiphilic peptides and investigated their antibacterial effect on MDR bacteria. A stable and potent dodecapeptide termed WRK-12 was identified, which exerts excellent activity against various pathogenic bacteria including carbapenems, colistin and tigecycline-resistant Gram-negative bacteria. Furthermore, WRK-12 displays low hemolytic activity and high salt or serum stability. Mechanical studies revealed that WRK-12 causes membrane damage and over-production of ROS through targeting LPS and bacterial-specific lipids. Collectively, the discovery of potent antimicrobial leads offers a novel therapeutic strategy to combat the increasing MDR bacteria.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/9/1398/s1, Table S1: Bacterial strains used in this study; Figure S1: HPLC spectra of engineered antimicrobial peptides on the basis of MP196; Figure S2: MS spectra of engineered antimicrobial peptides on the basis of MP196.
Author Contributions: Z.W. and Y.L. designed this study. Y.L. and J.S. performed all experiments. Y.L., J.S., Z.T., Y.J. and K.Y. analyzed the data. Y.L. and J.S. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.