Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)2 and Its Serum Degradation Product against Multidrug-Resistant Bacteria

Antimicrobial peptides can be used systemically, however, their susceptibility to proteases is a major obstacle in peptide-based therapeutic development. In the present study, the serum stability of p-BthTX-I (KKYRYHLKPFCKK) and (p-BthTX-I)2, a p-BthTX-I disulfide-linked dimer, were analyzed by mass spectrometry and analytical high-performance liquid chromatography (HPLC). Antimicrobial activities were assessed by determining their minimum inhibitory concentrations (MIC) using cation-adjusted Mueller–Hinton broth. Furthermore, biofilm eradication and time-kill kinetics were performed. Our results showed that p-BthTX-I and (p-BthTX-I)2 were completely degraded after 25 min. Mass spectrometry showed that the primary degradation product was a peptide that had lost four lysine residues on its C-terminus region (des-Lys12/Lys13-(p-BthTX-I)2), which was stable after 24 h of incubation. The antibacterial activities of the peptides p-BthTX-I, (p-BthTX-I)2, and des-Lys12/Lys13-(p-BthTX-I)2 were evaluated against a variety of bacteria, including multidrug-resistant strains. Des-Lys12/Lys13-(p-BthTX-I)2 and (p-BthTX-I)2 degraded Staphylococcus epidermidis biofilms. Additionally, both the peptides exhibited bactericidal activities against planktonic S. epidermidis in time-kill assays. The emergence of bacterial resistance to a variety of antibiotics used in clinics is the ultimate challenge for microbial infection control. Therefore, our results demonstrated that both peptides analyzed and the product of proteolysis obtained from (p-BthTX-I)2 are promising prototypes as novel drugs to treat multidrug-resistant bacterial infections.


Introduction
Infectious diseases are one of the main causes of death worldwide, particularly in developing countries. The increase in bacterial resistance to conventional antibiotics is a consequence of their widespread and uncontrolled use. Thus, the search for new therapeutic molecules to control microbial infections is highly important for tackling this challenge [1][2][3][4].
Antimicrobial peptides (AMPs) are a potential alternative treatment to traditional antibiotics owing to their efficacy, safety, enormous diversity, and broad-spectrum activity against a wide range of microorganisms, including oral bacteria [5,6], multidrug-resistant bacteria [7], fungi [8], and viruses [3]. To identify the serum degradation products of the peptides, mass spectrometry analyses were performed. After 5 min of incubation in serum, (p-BthTX-I)2 clearly produced two degradation products ( Figure 2A). Two different peptides were identified: one that had lost a single lysine residue ( Figure 2A, molecular mass segments in blue) and another that lost two lysine residues ( Figure 2A, molecular mass segments in red). Numbers in black are relative of the intact dimeric peptide.  2 were incubated with human serum for the indicated times. Analytical HPLC was performed on a Shimadzu (Kyoto, Japan) system using a C18 column (4.6 × 150 mm, Phenomenex) with a linear gradient of solvent B (5-95%) at 1 mL/min for 15 min (Solvent A: 0.045% trifluoroacetic acid (TFA) in water. Solvent B: 0.036% TFA in acetonitrile). Marked boxes in the figures show the same degradation product produced by both the peptides.
To identify the serum degradation products of the peptides, mass spectrometry analyses were performed. After 5 min of incubation in serum, (p-BthTX-I) 2 clearly produced two degradation products ( Figure 2A). Two different peptides were identified: one that had lost a single lysine residue (Figure 2A, molecular mass segments in blue) and another that lost two lysine residues (Figure 2A, molecular mass segments in red). Numbers in black are relative of the intact dimeric peptide. dimerization is swiftly initiated when the monomeric p-BthTX-I peptide is incubated in the culture medium [19], suggesting that only the dimer (p-BthTX-I)2 likely occurs in human serum. To identify the serum degradation products of the peptides, mass spectrometry analyses were performed. After 5 min of incubation in serum, (p-BthTX-I)2 clearly produced two degradation products ( Figure 2A). Two different peptides were identified: one that had lost a single lysine residue ( Figure 2A, molecular mass segments in blue) and another that lost two lysine residues ( Figure 2A, molecular mass segments in red). Numbers in black are relative of the intact dimeric peptide.  2 , black; (p-BthTX-I) 2 minus one Lys residue, blue; (p-BthTX-I) 2 minus two Lys residues, red; (p-BthTX-I) 2 minus three Lys residues, green; and (p-BthTX-I) 2 without four Lys residues, purple. After 10 min, the peptide that had lost two lysine residues was the major peptide product; however, the parent peptide and the other degradation product were still detected. Within 20 min of incubation, a peptide that had lost three lysine residues was identified ( Figure 2B, green numbers). After 30 min of incubation, a combination of peptide products having lost two, three, and four lysine residues could be observed ( Figure 2C). At later times (120 min), only the peptide product without the four lysine residues remained ( Figure 2D, purple numbers); this result was in agreement with the stable degradation product peak observed by HPLC. This peptide remained intact after 12 and 24 h; however, further degradation products were observed after 30 h of incubation (data not shown), highlighting the high stability of the degradation product without the four Lys residues.
Because the peptides p-BthTX-I and (p-BthTX-I) 2 exhibited specific antibacterial activities, we next tested the antibacterial and antifungal activities of the synthesized peptides against E. coli, Staphylococcus aureus, and C. albicans (Table 2) as well as a panel of multidrug-resistant clinical strains (Table 3). Of the total 20 bacterial strains tested, p-BthTX-I presented an antibacterial activity against S. epidermidis ATCC35984, S. aureus strains SA16, SA33, SA88 and SA90, Enterococcus faecium strains VRE16 and HSJRP8, Klebsiella pneumoniae NDM-1 and E. coli ATCC35218. The dimeric peptide was active against the same strains as its monomeric form and additionally showed an action against S. aureus ATCC25923, multidrug-resistant K. pneumoniae strains ATCC700603 and ATCC BAA1705 also against E. coli strains ATCC25922 and CA4, a commensal bacterium (Table 3). On the other hand, the serum degradation product, des-Lys 12 /Lys 13 -(p-BthTX-I) 2 , exhibited superior antimicrobial effect against S. aureus strains ATCC25923, SA16 and SA33, E. faecium strains VRE16 and HSJRP8, K. pneumoniae ATCC BAA1705 and E. coli strains ATCC35218 and CA4 (Table 3).
Next, the capacity of the peptides to eradicate biofilms formed by S. epidermidis was evaluated. The result showed that both peptides were able to degrade the biofilms formed by S. epidermidis ATCC35984 with similar effectiveness (78.6% and 80% for (p-BthTX-I) 2 and des-Lys 12 /Lys 13 -(p-BthTX-I) 2 , respectively; Figure 4).  Because the peptides p-BthTX-I and (p-BthTX-I)2 exhibited specific antibacterial activities, we next tested the antibacterial and antifungal activities of the synthesized peptides against E. coli, Staphylococcus aureus, and C. albicans (Table 2) as well as a panel of multidrug-resistant clinical strains (Table 3). Of the total 20 bacterial strains tested, p-BthTX-I presented an antibacterial activity against S. epidermidis ATCC35984, S. aureus strains SA16, SA33, SA88 and SA90, Enterococcus faecium strains VRE16 and HSJRP8, Klebsiella pneumoniae NDM-1 and E. coli ATCC35218. The dimeric peptide was active against the same strains as its monomeric form and additionally showed an action against S. aureus ATCC25923, multidrug-resistant K. pneumoniae strains ATCC700603 and ATCC BAA1705 also against E. coli strains ATCC25922 and CA4, a commensal bacterium (Table 3). On the other hand, the serum degradation product, des-Lys 12 /Lys 13 -(p-BthTX-I)2, exhibited superior antimicrobial effect against S. aureus strains ATCC25923, SA16 and SA33, E. faecium strains VRE16 and HSJRP8, K. pneumoniae ATCC BAA1705 and E. coli strains ATCC35218 and CA4 (Table 3). Table 2. Biological activities of the synthetic peptides.    2 , and des-Lys 1 /Lys 2 -(p-BthTX-I) 2 to the RT of peaks of the stable serum degradation product of (p-BthTX-I) 2 . Samples were eluted at 5 min and 3 h. Analytical HPLC was performed on a Shimadzu system using a C18 column (4.6 × 150 mm, Phenomenex) with a linear gradient of solvent B (5-95%) at 1 mL/min for 30 min (Solvent A: 0.045% trifluoroacetic acid (TFA) in water. Solvent B: 0.036% TFA in acetonitrile). Marked box indicates the peak of des-Lys 12 /Lys 13 -(p-BthTX-I) 2 and the stable degradation peptide produced.    To confirm the bactericidal activity of (p-BthTX-I) 2 and des-Lys 12 /Lys 13 -(p-BthTX-I) 2 against the planktonic form of S. epidermidis ATCC35984, time-kill assays were performed. The results showed that at all concentrations tested (1×, 2×, and 4× minimum inhibitory concentration, MIC) of (p-BthTX-I) 2 ( Figure 5A) and des-Lys 12 , Lys 13 -(p-BthTX-I) 2 ( Figure 5B) were capable of eradicating the bacteria in the initial minutes of incubation, as evidenced by the log 10 Colony Forming Units (CFU) /mL value differing from that of the growth control at time 0. Next, the capacity of the peptides to eradicate biofilms formed by S. epidermidis was evaluated. The result showed that both peptides were able to degrade the biofilms formed by S. epidermidis ATCC35984 with similar effectiveness (78.6% and 80% for (p-BthTX-I)2 and des-Lys 12 /Lys 13 -(p-BthTX-I)2, respectively; Figure 4). To confirm the bactericidal activity of (p-BthTX-I)2 and des-Lys 12 /Lys 13 -(p-BthTX-I)2 against the planktonic form of S. epidermidis ATCC35984, time-kill assays were performed. The results showed that at all concentrations tested (1×, 2×, and 4× minimum inhibitory concentration, MIC) of (p-BthTX-I)2 ( Figure 5A) and des-Lys 12 , Lys 13 -(p-BthTX-I)2 ( Figure 5B) were capable of eradicating the bacteria in the initial minutes of incubation, as evidenced by the log10 Colony Forming Units (CFU) /mL value differing from that of the growth control at time 0. The result showed that both peptides were able to degrade the biofilms formed by S. epidermidis ATCC35984 with similar effectiveness (78.6% and 80% for (p-BthTX-I)2 and des-Lys 12 /Lys 13 -(p-BthTX-I)2, respectively; Figure 4). To confirm the bactericidal activity of (p-BthTX-I)2 and des-Lys 12 /Lys 13 -(p-BthTX-I)2 against the planktonic form of S. epidermidis ATCC35984, time-kill assays were performed. The results showed that at all concentrations tested (1×, 2×, and 4× minimum inhibitory concentration, MIC) of (p-BthTX-I)2 ( Figure 5A) and des-Lys 12 , Lys 13 -(p-BthTX-I)2 ( Figure 5B) were capable of eradicating the bacteria in the initial minutes of incubation, as evidenced by the log10 Colony Forming Units (CFU) /mL value differing from that of the growth control at time 0.

Discussion
In a previous study, Santos-Filho et al. synthesized and characterized the peptide p-BthTX-I (KKYRYHLKPFCKK) and its disulfide-linked dimeric form (p-BthTX-I)2 [19]. These peptides exhibited antimicrobial activity against E. coli ATCC25922 (MIC values of 16 and 4 µM for p-BthTX-I and (p-BthTX-I)2, respectively) and S. aureus ATCC25923 (MIC values of 64 and 32 µM for p-BthTX-I and (p-BthTX-I)2, respectively) and were nontoxic to C. albicans ATCC18804, erythrocytes, epithelial cells, and macrophages, indicating a potential specificity against prokaryotic cells. Furthermore, replacement of cysteine to a serine in the peptide attenuated its antibacterial activity. Thus, it was concluded that dimerization was crucial for the antimicrobial activity of p-BthTX-I and that the

Discussion
In a previous study, Santos-Filho et al. synthesized and characterized the peptide p-BthTX-I (KKYRYHLKPFCKK) and its disulfide-linked dimeric form (p-BthTX-I) 2 [19]. These peptides exhibited antimicrobial activity against E. coli ATCC25922 (MIC values of 16 and 4 µM for p-BthTX-I and (p-BthTX-I) 2 , respectively) and S. aureus ATCC25923 (MIC values of 64 and 32 µM for p-BthTX-I and (p-BthTX-I) 2 , respectively) and were nontoxic to C. albicans ATCC18804, erythrocytes, epithelial cells, and macrophages, indicating a potential specificity against prokaryotic cells. Furthermore, replacement of cysteine to a serine in the peptide attenuated its antibacterial activity. Thus, it was concluded that dimerization was crucial for the antimicrobial activity of p-BthTX-I and that the result of the monomeric form was due to the fast dimerization of the peptide in the culture medium [19]. The effects of dimerization on the antimicrobial peptide remain unclear, although previous studies have indicated increases or decreases in antimicrobial activity [7,[20][21][22][23].
Concerns have been raised regarding antimicrobial peptide administration into the circulatory system; in vitro assays have suggested that the peptides are usually less effective, owing to a lack of resistance against serum proteases, which causes proteolytic degradation and low activity under the physiological conditions [24,25]. Because of these limitations, development of antimicrobial peptides for clinical usage has been restricted [11]. The development of different drug delivery systems could aid in administering antimicrobial peptides, enhancing their half-lives, maintaining and improving activities against specific targets, and decreasing toxicity.
Dimeric peptides are used as an alternative approach for enhancing half-life and increasing biological activity because they are less susceptible to protease degradation than monomeric peptides [19,22,26]. Thus, serum stability assays were performed to analyze proteolysis of p-BthTX-I and (p-BthTX-I) 2 in human blood. Analytical HPLC analyses indicated that after incubation with human serum for 30 min, both peptides were completely degraded. In addition, the stable product des-Lys 12 /Lys 13 -(p-BthTX-I) 2 was characterized.
Although antimicrobial peptides could be used systemically, susceptibility to proteolytic degradation by proteases is a significant hindrance to the development of peptide-based therapy [24,27]. Serum exhibits extensive protease and peptidase activity [28], and peptidomic studies have shown diverse peptidases in human blood [29]. There are three major classes of peptidases in blood: (1) aminoand (2) carboxyexopeptidases, which cleave at the amino and carboxyl termini, respectively, and (3) endopeptidases, which cleave internal peptide bonds [30]. Based on this classification and the results presented herein, (p-BthTX-I) 2 is likely degraded by blood carboxypeptidase B, which specifically hydrolyzes C-terminal lysine and arginine residues (lysine carboxy-exopeptidases) [31].
The antimicrobial activity of the synthesized peptides, including des-Lys 12 /Lys 13 -(p-BthTX-I) 2 , the primary degradation product, was evaluated ( Table 2). Similar to (p-BthTX-I) 2 [19], all of the peptides exhibited antibacterial activity. Furthermore, they do not exhibited antifungal activity against C. albicans or toxicity against erythrocytes, suggesting that these peptides have promising therapeutic potential owing to their specificity for prokaryotic cells.
Additionally, circular dichroism (CD) spectroscopy was performed for all the peptides to evaluate the association between their antibacterial activity and structure. The peptides had similar spectra and had predominantly random coil content (data not shown). Our previous work showed that the parent peptide (p-BthTX-I) 2 does not interact with membranes [19] (a common trait for peptides with high random coil content). Our data indicated that the peptide could not act on membrane mimetics as their mechanism of action (data not shown); this has been discussed previously [19]. Therefore, a detailed assessment of this peptide could promote a new class of antimicrobial compounds.
There are several concerns regarding the serum stability of (p-BthTX-I) 2 . After administration into the circulatory system, the antimicrobial effects of the peptide against bacteria may be faster than its degradation rate. Moreover, despite degradation of the dimeric peptide, the generated product also exhibited antibacterial activity.
P-BthTX-I, (p-BthTX-I) 2 , and des-Lys 12 /Lys 13 -(p-BthTX-I) 2 exhibited antimicrobial activity against a variety of bacteria, including multidrug-resistant clinical strains (Table 3). Importantly, antimicrobial activity against resistant bacteria was analyzed using cation-adjusted MH broth. Development of antimicrobial peptides has been hindered by several problems, such as salt sensitivity, as antimicrobial activity can be altered by salts (inactivation or increased MIC) [32][33][34][35][36]; thus, this step was an important consideration for testing the peptide activity. Of the bacterial strains tested, the panel of multidrug-resistant clinical strains was critical for determining the therapeutic potential of these peptides. S. aureus SA16, SA33, SA88, and SA90 are methicillin-resistant S. aureus (MRSA) strains isolated from infected Brazilian patients and belong to the widespread clones ST5/105SCCmecII [37]. SA33 is also resistant to tigecycline, whereas SA88 is also daptomycin heteroresistant. MRSA has become one of the most important nosocomial pathogens worldwide, capable of causing a variety of hospital infections [38]. Hospital infections have been steadily increasing over the last few decades and are now mainstream due to the emergence of drug-resistant bacterial strains [39]. Among the most prevalent bacteria causing nosocomial infections, K. pneumoniae, can lead to serious infections, including urinary tract infections, hospital-acquired pneumonia, intra-abdominal infections, wound infections, and primary bacteremia [40]. (p-BthTX-I) 2 was also active against K. pneumoniae ATCC700603, ATCCBAA1705, and NDM-1 strains, with the last two capable of producing β-lactamases [41]. K. pneumoniae ATCCBAA1705 produces K. pneumoniae carbapenemase (KPC), whereas NDM-1 produces a New Delhi Metallo-β-lactamase 1 (NDM-1). Moreover, K. pneumoniae ATCC700603 produces SHV-18, a β-lactamase that renders this strain resistant to several β-lactam antibiotics.
The peptides also were active against E. faecium and S. epidermidis. Normally, E. faecium has no adverse effects on the host, however, it can cause endocarditis, bacteremia, urinary tract infections, and meningitis, primarily in immunocompromised patients [42]. Enterococcus spp. are particularly relevant to the medical community owing to their incidence of antibiotic resistance [43]. E. faecium VRE16 is a high-risk, vancomycin-resistant enterococci clone of ST412 isolated from nosocomial infected patients worldwide [44], whereas the HBSJRP8 strain is daptomycin resistant.
S. epidermidis is primarily known as an innocuous, commensal bacterium on human skin. However, antibiotic-resistant S. epidermidis, mainly methicillin-resistant (MRSE), has gained increasing medical relevance [45,46]. Moreover, it has emerged as a major nosocomial pathogen associated with infections of implanted medical devices because of its biofilm formation properties [47]. Staphylococcal biofilms are characterized as a multicellular aggregate surrounded by a self-produced extracellular matrix consisting of proteins, polysaccharides, and extracellular DNA (eDNA; originating from the bacteria autolysis) [48,49]. The biofilm-forming cells can adhere to a variety of surfaces and are the major cause of nosocomial infections that colonize biomedical devices, such as respirators, catheters, prosthetic heart valves, and orthopedic devices [50,51]. Biofilms are a defense mechanism within bacteria, rendering them up to 1000 times more resistant to antimicrobial agents than their planktonic counterparts [52]. High levels of resistance can be due to delayed penetration of antimicrobial agents and changes in the metabolic rates of the microorganisms [53].
Biofilm eradication activity was analyzed against S. epidermidis ATCC35984, which is known to produce strong biofilms, isolated from a patient with intravascular catheter-associated sepsis [54,55] ( Figure 4). Our data indicated that both (p-BthTX-I) 2 and des-Lys 12 /Lys 13 -(p-BthTX-I) 2 were effective against this strain (MIC of 16 and 32 µmol/L, respectively), suggesting that both molecules have therapeutic potential for developing new drugs.

Electrospray Mass Spectrometry
To confirm the identity of the peptides, diluted samples were analyzed by direct infusion mass spectrometry using an amaZon ion trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA) coupled to a syringe pump (2.5-10 µL/min) in a positive electrospray ionization mode (ESI).

Reverse Phase Chromatography
Crude peptides were purified by semi-preparative HPLC on a Shimadzu system (Tokyo, Japan) using a C18 reversed phase column (10 × 250 mm, Phenomenex, Torrance, CA, USA). Solvent A contained 0.045% trifluoroacetic acid (TFA) in water. Elution was achieved using a linear gradient (5-35%) of solvent B (0.036% TFA in acetonitrile) for 120 min at 5 mL/min. The purity of the peptides was determined by analytical HPLC on a Shimadzu system using a C18 column (4.6 × 150 mm, Phenomenex, Torrance, CA, USA) with a linear gradient of solvent B (5-95%) for 30 min at 1 mL/min. Liquid chromatography-mass spectrometry (LC/MS) was performed using a C18 column (2.0 × 30 mm, Shimadzu) attached to analytical HPLC and an amaZon ion trap mass spectrometer (Bruker Daltonics) using ESI. Peptides were eluted using a linear gradient of solvent B (5-95%) for 15 min at 0.2 mL/min.

Serum Stability Assay
Peptide stability assays were performed in diluted serum as previously described [24,25] with minor modifications. First, 2 mL of 25% human male serum was centrifuged at 3000× g for 10 min; the supernatant was collected and incubated for 15 min at 37 • C. Next, peptides were added to the serum to a final concentration of 100 µM. Then, 200 µL of samples were collected at different time points during the assays, which were performed in duplicate. Analyses were performed by LC/MS and analytical HPLC, as described above.

Circular Dichroism Spectroscopy
CD spectra of peptides were recorded from 190 to 250 nm at room temperature with a J-815CD spectrophotometer (Jasco Co., Tokyo, Japan) under continuous nitrogen flush using 1-mm path length quartz cuvettes. To compare the secondary structures of the peptides, spectra were obtained in an aqueous solution (phosphate buffer saline, PBS), in a secondary structure-inducing solvent (trifluoroethanol, TFE), and in lysophosphatidylcholine (LPC). The peptide concentration was 60 µM. CD spectra were typically the average of six scans, measured in millidegrees.

Hemolysis Assay
Peptide hemolysis assays were performed as previously described by Castro et al. [58]. Briefly, freshly prepared human red blood cells (RBCs) were washed three times with 0.01 M Tris-HCl (pH 7.4) containing 0.15 M NaCl (Tris-saline). A suspension of 1% (v/v) erythrocytes was made with packed RBCs resuspended in Tris-saline. Peptides were dissolved in Tris-saline at an initial concentration of 128 mM and serially diluted in the same buffer to determine the concentration that triggered 50% hemolysis (HC 50 ). As the positive control (100% lysis), 1% (v/v) Triton X-100 solution was used. After 1 h of incubation at 37 • C, the samples were centrifuged at 3000× g for 5 min. Then, 100 µL aliquots of the supernatant were transferred to 96-well microplates, and the absorbance was determined at 405 nm. The assay was performed in triplicates.

Fluorescence Spectroscopy
Fluorescence data acquisition was performed using an Eclipse spectrofluorometer (Varian Cary, Agilent Technologies, Santa Clara, CA, USA). The excitation was 276 nm to irradiate tyrosine groups, and the emission spectra were measured from 294 to 365 nm. Fluorescence spectra were obtained using peptide concentrations of 30 µM to a final volume of 600 µL. For studies of peptide-micelle interactions, titration of the peptides was performed in the zwitterionic detergent LPC. The peptide concentrations ranged from 0 to 10 mM in Tris-HCl, 0.01 M NaCl, pH 7.4. Fluorescence intensity and maximum emission wavelength data were obtained.

In Vitro Evaluation of the Antimicrobial Activity
The minimum inhibitory concentrations (MIC) were determined following the Clinical and Laboratory Standards Institute (CLSI) recommendations [59]. Antibacterial and antifungal activity tests were performed using the microdilution method. For initial screening, peptides were tested against C. albicans ATCC18804, E. coli ATCC25922, and S. aureus ATCC25923. Briefly, bacterial cells in MH broth (80 µL aliquots containing 1.5 × 10 7 CFU) were incubated with synthetic peptides (serial dilution from 512 to 1 µM) dissolved in Milli-Q water. After incubation for 24 h at 37 • C, the microtiter plates were analyzed visually by addition of resazurin. For the antifungal assays, the culture medium was RPMI 1640 buffered with L-glutamine (pH 7.2), 0.165 M morpholinepropanesulfonic acid (MOPS), and 2% glucose. Cell suspensions (final concentration of 1 × 10 3 -2.5 × 10 3 CFU/mL) were inoculated on a microdilution plate previously prepared with serially diluted synthetic peptides (128-1 µM). The plates were incubated for 48 h at 37 • C. Each assay was performed in triplicates. Additionally, amphotericin B and fluconazole were used as the control drugs. MIC was defined as the lowest concentration of peptide at which no growth was detected.
After confirming antibacterial activity, the peptides were tested against gram-positive and gram-negative multidrug-resistant bacteria, including strains of clinical origin. The peptides, p-BthTX-I, des-Lys 12 /(p-BthTX-I) 2 , and Lys 13 -(p-BthTX-I) 2 were serially diluted (512-1 µM) in cation-adjusted BBL MH II broth (CAMHB; Becton, Dickson and Co., Sparks, MD, USA) as described by CLSI. Bacteria at 5.0 × 10 5 CFU/mL were added to the media and incubated for 24 h at 37 • C. The microtiter plates were analyzed visually, and MIC was defined as the lowest concentration that completely inhibited bacterial growth. Assays were performed in duplicates, and sterile broth was used as the negative control. To determine the minimal bactericidal concentration (MBC), 100 µL from each well of the MIC assay plates that inhibited bacterial growth were sub-cultured onto MH agar (MHA) plates. The plates were incubated for 24 h at 37 • C, and MBC was defined as the lowest concentration of the peptides that resulted in 99.9% bacterial death. If the ratio of MBC/MIC was >4, the activity of the peptide was considered bacteriostatic, as described previously [60].

Biofilm Eradication
The biofilm eradication abilities of (p-BthTX-I) 2 and des-Lys 12 /Lys 13 -(p-BthTX-I) 2 were evaluated as previously described [61]. Briefly, S. epidermidis ATCC35984, known to produce biofilms, was cultured for 18 h on brain heart infusion broth (BHI; KASVI, Curitiba, PR, Brazil) supplemented with 0.75% glucose (w/v). Bacterial suspension was adjusted to an OD 600 of 1 and then diluted to 1:40 in the same broth. Next, 200 µL of the bacterial dilution was added per well to a 96-well plate and incubated for 24 h at 37 • C. After bacterial adhesion, the plate was washed three times in PBS (pH 7.4), and the bacteria cells were incubated with either only the media (positive control) or with 512 µM peptides, des-Lys 12 /(p-BthTX-I) 2 , and Lys 13 -(p-BthTX-I) 2 , at 37 • C for 24 h. The samples were then washed in PBS before staining with crystal violet (0.2% w/v), and peptide activities were evaluated at 595 nm in a microplate reader (Polaris, Celer, Belo Horizonte, Minas Gerais, Brazil). A total of 16 experimental replicates were performed for each condition. One-way ANOVA was used to compare the absorbance values of the peptide treatments with the positive control. p < 0.05 was considered statistically significant.

Time-Kill Kinetics
The bactericidal activities of (p-BthTX-I) 2 and des-Lys 12 /Lys 13 -(p-BthTX-I) 2 against planktonic cells of S. epidermidis ATCC35984 were analyzed by a time-kill assay, as described previously [62]. Briefly, a bacterial suspension cultured for 18 h in BHI broth was adjusted with sterile 0.9% NaCl to 1.0 McFarland turbidity standard (approximately 3.0 × 10 8 CFU/mL). The standardized bacterial suspension was diluted to 1:5 in CAMHB to approximately 6.0 × 10 7 CFU/mL. Next, 100 µL of the adjusted bacterial suspension was added to 10 mL of CAMHB containing 1×, 2×, and 4× MIC concentrations of des-Lys 12 /(p-BthTX-I) 2 or Lys 13 -(p-BthTX-I) 2 , and the final concentration was approximately 6.0 × 10 5 CFU/mL. Immediately after inoculation, tubes were vortexed, and 100 µL was eluted from each tube and serially diluted in sterile 0.9% NaCl. Dilutions were plated on BHI agar (six replicates of 15 µL each) and incubated for 24 h at 37 • C for the zero time point. Tubes were incubated at 37 • C under constant agitation, and 100 µL aliquots were eluted after 3 and 6 h. Serial dilutions were plated on BHI agar and incubated for 24 h at 37 • C. Colonies were counted, and the results were recorded as log 10 CFU/mL. A decrease in value of ≥3 log 10 CFU/mL was considered bactericidal. Bacteria cultured in the absence of the peptides were used as the positive control and sterile broth was used as the negative control. The experiment was performed twice (total of two biological replicates).

Conclusions
In this study, the serum stability of the peptide (p-BthTX-I) 2 was investigated. Encouragingly, the stable degradation product des-Lys 12 /Lys 13 -(p-BthTX-I) 2 exhibited activity similar to that of the dimeric form (p-BthTX-I) 2 . These data suggest that after proteolysis of the parent peptide, the product retains antibacterial activity. In particular, des-Lys 12 /Lys 13 -(p-BthTX-I) 2 is a potential peptide for antibacterial drug design because shorter peptides are cheaper to produce synthetically. Therefore, these peptides can be used as models for new drugs to target multidrug-resistant infections worldwide.