Wongchai et al.

Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Annals of Clinical Microbiology
https://doi.org/10.1186/s12941-024-00701-7
and Antimicrobials

RESEARCH Open Access

Dual-function antimicrobial-antibiofilm
peptide hybrid to tackle biofilm-forming
Staphylococcus epidermidis
Mathira Wongchai1, Saharut Wongkaewkhiaw2, Sakawrat Kanthawong3, Sittiruk Roytrakul4 and
Ratchaneewan Aunpad1*

Abstract
Background Due to their resistance and difficulty in treatment, biofilm-associated infections are problematic among
hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have
recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of
biofilm inhibition and eradication.
Methods A novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm
peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif
(KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions
5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm
and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using
crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of
peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K,
was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed
using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability
were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser
scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined
using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its
preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area.
Results BiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S.
epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular
polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum
antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its
activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed
an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed,

*Correspondence:
Ratchaneewan Aunpad
[email protected]
Full list of author information is available at the end of the article

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Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 2 of 19

demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent
the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area.
Conclusions Due to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to
combat biofilm infections, particularly those of biofilm-forming S. epidermidis.
Keywords Antibiofilm peptide, Staphylococcus epidermidis, Biofilm, Antibacterial activity, Lipid binding motif

Background whether planktonic (free-floating) or embedded within

Hospital-associated infections (HAIs), also referred to biofilms [10]. Moreover, a significant advantage of pep-
nosocomial infections, pose a significant global public tides is their low propensity to induce resistance at sub-
health concern. Each year, there were around 40,000 hos- inhibitory concentrations, likely due to their mechanistic
pitalized patients’ death due to these infections and an differences from conventional antibiotics [11, 12]. These
escalating mortality was observed both in developing and peptides exert their antibiofilm effects through diverse
developed countries [1, 2]. More than half (50–70%) of mechanisms which vary with the concentration of the
HAIs are caused by the development of biofilms on med- peptide. At inhibitory concentrations or higher, peptides
ical devices which are increasingly used in many aspects possess the capacity to directly kill bacteria before bio-
of patient care [2–4]. In healthcare settings, the presence film formation, within the biofilm matrix, or during the
of biofilm-related infections is significant concern since detachment process. At subinhibitory concentrations,
they can compromise the effectiveness of antibiotic and the peptides exhibit distinct mechanisms which include
immune therapeutics [5]. Thereby, they contribute to the interference with gene expression, bacterial adhesion, or
problem of antibiotic resistance and the persistence of host immune response [10, 13]. This variety of mecha-
infections [6]. nisms makes these peptides highly effective against bac-
Infections caused by Staphylococcus epidermidis are a terial biofilms at various stages of development.
significant global concern, as they account for approxi- Rational design of antimicrobial peptides using
mately 80% of infections associated with medical devices sequence optimization and modification has been widely
[3]. Normally, S. epidermidis is considered a non-patho- recognized since it can significantly enhance effective-
genic commensal member of human skin flora, coloniz- ness, stability, and specificity [14]. In this study, various
ing the skin with relatively non-invasive mechanisms, strategies, including peptide hybridization and amino
and expressing few virulence factors. However, it has a acid substitution, were exploited to optimize design of
remarkable capability to attach to and colonize the sur- the antibiofilm peptide. The hybrid peptide R-FV-I16,
faces of medical devices, forming biofilms which are with robust antimicrobial and antibiofilm activities, was
associated with subsequent infections [7]. The distinctive obtained via the insertion of a non-functional sequence
features characterizing S. epidermidis biofilms include (RR7) along with an antibiofilm sequence (FV7) [15]. The
the production of polysaccharide intracellular adhesin substitution of leucine and lysine in Temporin 1Tb, yield-
(PIA) by the intracellular adhesion (ica) gene locus, and ing the peptide TB_L1FK, has demonstrated an increase
biofilm regulatory control through the accessory gene in antimicrobial activity and concurrent inhibition of bio-
regulator (agr) quorum-sensing system which is con- film formation [16].
served across various staphylococcal species [8]. In this research, a novel and highly effective antibiofilm
The treatment of biofilm infections is often challenging, peptide was designed by combining various strategies
and antibiotic combinations are typically recommended. including a template-assisted approach, peptide hybrid-
However, higher concentrations of each antibiotic than ization and sequence modification. A conserved sequence
against planktonic cells are generally required [9]. Regret- derived from a well-established 18 α-helical antibiofilm
tably, eradication of device-related infections typically peptide was hybridized with a previously described lipid-
requires removal of the device and there is a high risk of binding motif, the KILRR motif [17]. The inclusion of a
reinfection, along with patient trauma and additional cost lipid-binding motif represents an intriguing strategy for
[4]. For these reasons, there is necessary to develop novel increasing the peptide’s affinity for bacterial membrane
antibiofilm agents as well as improved strategies to treat and lipid constituents within the biofilm matrix. This
biofilm-associated infections. novel peptide serves as a basic scaffold for the develop-
Antibiofilm peptides provide a promising approach for ment of a series of derivative peptides by amino acid sub-
combating biofilm-causing bacterial infections, with need stitution. Among them, BiF2_5K7K, a lysine-substituted
particularly focused on infections of indwelling medi- hybrid peptide, exhibited remarkable antibiofilm and
cal devices. One noteworthy feature of antibiofilm pep- antimicrobial activities, particularly against S. epider-
tides is their versatility in regard to targeting of bacteria, midis biofilm-forming strains, with minimal hemolytic
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 3 of 19

effect and cytotoxicity. Even at subinhibitory concentra- Peptide synthesis and preparation
tions, BiF2_5K7K was able to prevent the formation of S. The peptides were generated using solid-phase peptide
epidermidis biofilm via modulating biofilm-related genes synthesis with 9-fluorenylmethoxycarbonyl (Fmoc) as
expression. Additionally, BiF2_5K7K demonstrated its trifluoroacetate salts and amidation at the C-terminus
ability to prevent biofilm formation on catheter surfaces. (China Peptides, China). Reverse phase high-perfor-
This work marked a significant step toward the develop- mance liquid chromatography (RP-HPLC; >98% purity)
ment of novel antibiofilm agents, which may have impli- and mass spectrometry were employed to confirm the
cations for combating biofilm infections, particularly identity and purity of the peptide sequences, respectively.
those associated with medical devices. The peptides were dissolved in sterile deionized water
and kept at -20°C until used.
Materials and methods
Bacterial strains and growth conditions Biofilm formation capacity of S. epidermidis ATCC 35984
The biofilm-forming bacteria, including S. epidermi- The capability of S. epidermidis ATCC 35984 to form
dis ATCC 35984, Staphylococcus aureus ATCC 25923, biofilm was evaluated by crystal violet (CV) staining
Bacillus cereus ATCC 11778, Listeria monocytogenes as described previously [18]. Briefly, bacterial suspen-
10403s, Pseudomonas aeruginosa ATCC 27853, Salmo- sions derived from an overnight culture were harvested
nella Typhimurium ATCC 13311, Escherichia coli ATCC and centrifuged at 2,500 × g for 5 min to collect the pel-
25922 and Acinetobacter baumanii MT strain, were let. Then, TSB was used to resuspended and diluted the
used. All bacterial strains were grown on tryptic soy agar pellet to achieve a concentration of 106 CFU/mL at opti-
(TSA; BD Bacto™, USA) except E. coli and L. monocyto- mal density (OD) of 620 at 0.05. These diluted suspen-
genes which were cultured on Luria agar (LA) and TSA sions were subsequently dispensed into 96-well plates
supplemented with 0.6% Yeast Extract (TSAYE), respec- and statically incubated for 24 h at 37°C. Following the
tively, at 37°C overnight. Subsequently, isolated colonies incubation period, both the culture media and plank-
were inoculated and cultured in 5 mL of tryptic soy broth tonic cells were discarded, and the biofilm in each well
(TSB; BD Bacto™, USA), Luria Broth (LB), or TSB sup- was washed thrice with 0.85% NaCl. Subsequently, the
plemented with 0.6% Yeast Extract (TSBYE) at 37°C for biofilms were fixed using 95% ethanol and stained with a
16–18 h. 0.1% (v/v) solution of CV solution (Sigma-Aldrich, USA)
for 15 min. Excess dye was removed, and each well was
Peptide design and in silico characteristics washed thrice with 0.85% NaCl before resuspended in
Using a lesson learned from peptides designed by a 95% ethanol to dissolve the bound dye. Absorbance was
template-assisted approach, a novel antibiofilm peptide, determined at 570 nm using a Multiskan SkyHigh micro-
BiF, was developed from the alignment of conserved plate reader (Thermo Scientific™, Singapore). Wells con-
sequences of 18 α-helical antibiofilm peptides obtained taining only TSB medium without bacteria served as the
from APD3 (Antimicrobial Peptide Database, retrieved negative control. All experiments were conducted with
on 7 August 2019). To further enhance its antibiofilm six technical replicates and repeated independently three
activity, a lipid binding motif (or KILRR motif [17]) was times. The biofilm-forming ability of the bacteria was
hybridized at the C-terminus of BiF and transformed categorized into four groups based on the OD570 value:
into hybrid BiF2 peptide. In accord with the results of no biofilm production (OD570≤ ODc), weak biofilm pro-
helical wheel projections and 3D structures, BiF2 was duction (ODc<OD570≤ 2 × ODc), moderate biofilm pro-
subsequently modified by substituting in one and two duction (2 × ODc<OD570≤4 × ODc), and strong biofilm
hydrophobic amino acids at positions 5 and 7 with lysines production (4 × ODc<OD570). The mean OD ± three
(L5K, L7K), positively charged amino acid, to obtain two standard deviations (SD) of the OD value of the negative
hybrid derivatives, BiF2_5K and BiF2_5K7K, respec- control was defined as cut-off OD (ODc).
tively. Peptide sequences were in silico analysed by APD3:
Antimicrobial Peptide Calculator and Predictor (https:// Biofilm-formation inhibitory activity
aps.unmc.edu/prediction). Both hydrophobicity (<H>) The biofilm inhibitory activity of peptide derivatives was
and hydrophobic moment (<μH>), as well as helical assessed by modified crystal violet (CV) staining [19].
wheel projection, were determined using the HeliQuest Briefly, the pellet of S. epidermidis (ATCC 35984) was
web server (https://heliquest.ipmc.cnrs.fr/). Moreover, collected through centrifugation of bacterial suspensions
I-TASSER (https://zhanglab.dcmb.med.umich.edu/I- grown overnight at 2,500 × g for 5 min. Subsequently,
TASSER/) was employed to predict the 3D structure of the pellet was resuspended and diluted with TSB to a
these peptides. final concentration of 106 CFU/mL. Then, 50 µL of these
diluted suspensions was transferred to 96-well micro-
plates containing same volume of peptide derivatives
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 4 of 19

in 10 mM phosphate buffer saline (PBS; Caisson Labs, An equal volume of 2% hRBCs and peptide at concen-
USA), with concentrations ranging from 0.98 to 250 µg/ tration of 0.98–250 µg/mL in 10 mM PBS mixtures
mL. Vancomycin was used as positive control while TSB were incubated for 1 h at 37°C. Following incubation,
medium without bacteria was used as negative con- the suspension was centrifuged, then supernatants were
trol. After 24 h of incubation and reaching static growth collected and transferred to 96-well microplates for mea-
at 37°C, culture media as well as planktonic cells were surement of absorbance at OD of 405 nm (OD405). This
removed, then the biofilm in each well was washed thrice study was performed in accordance with the Thammasat
with 0.85% NaCl. For CV staining, biofilms were fixed University Ethics Committee (COA No. 066/2562). Writ-
with 95% ethanol and stained with a 0.1% (v/v) CV solu- ten informed consent was obtained from all volunteers.
tion for 15 min. After removal of excess dye, each well
was washed thrice with 0.85% NaCl and resuspended Biofilm cell viability
in 95% ethanol to dissolve bound dye. Absorbance was The biofilm cell viability after treated with hybrid peptide
determined at 570 nm using a Multiskan SkyHigh micro- BiF2_5K7K was examined by 3-(4,5-Dimethylthiazol-
plate reader (Thermo Scientific™, Singapore). All experi- 2-yl)-2,5-Diphenyltetrazolium bromide or MTT colori-
ments were done in six technical replicates and repeated metric assays [16]. Bacterial suspensions (106 CFU/mL
independently three times. Minimum biofilm inhibitory in TSB) and peptide at concentration ranging from 0.98
concentrations at 50% and 90% (MBIC50 and MBIC90) to 250 µg/mL in 10 mM PBS were incubated in 96-well
refers to concentrations of the peptide at which the inhi- microtiter plates at 37°C for 24 h. After incubation,
bition of biofilm was at least 50% and 90%, respectively supernatant was discarded, followed by the addition of
[19, 20]. 0.4 mg/mL MTT (Sigma-Aldrich, USA) in 10 mM PBS
to each well and incubated for 3 h at 37°C. Subsequently,
Antimicrobial activity supernatant was removed and dimethyl sulfoxide
The minimal inhibitory concentrations (MICs) of pep- (DMSO; Fisher BioReagents™, UK) was added to dissolve
tide derivatives versus planktonic bacteria were assessed the formed formazan crystals in wells. At a wavelength of
using a modification of the broth microdilution assay pre- 570 nm, the optical densities of the solutions were then
viously described in the National Committee for Clinical determined by microplate reader.
Laboratory Standards (NCCLS) [21]. In brief, bacteria at
mid-logarithmic growth were collected by centrifugation Biofilm eradication activity
at 2,500 × g for 5 min. Subsequently, the pellet was resus- The eradication capacity of BiF2_5K7K against pre-
pended and adjusted in Mueller-Hinton Broth (MHB; formed biofilm was examined as previously described
BD Bacto™, USA) to an OD620 of 0.05. Fifty µL of bacte- with some modifications [22, 23]. In brief, suspensions of
rial suspensions was transferred to 96-well microplates. S. epidermidis with a final density of 106 CFU/mL were
Later, 50 µL of peptide derivatives at 0.98–250 µg/mL in added to 96-well microplates, then covered with 96-peg
10 mM PBS, was added to wells and incubated for 24 h at lids (Nunc™ Immuno TSP Lids, Denmark). Following
37°C with continuous shaking at 220 rpm. After incuba- 24 h of incubation at 37°C, the peg lids were washed with
tion, the minimum concentration of peptide that resulted 0.85% NaCl to remove culture media and non-adherent
in no bacterial growth observed by visible inspection cells. Afterwards, the biofilms adherent to peg lids were
after 24 h of incubation was defined as the MIC [19]. The incubated at 37°C for 24 h with different concentrations
experiments were done in three technical replicates and of peptide derivatives. The peg lids were subsequently
repeated independently. washed and placed on new plates containing PBS. The
To determine minimal bactericidal concentrations biofilms on the lid were removed using a water bath soni-
(MBCs) of the peptides, 50 µL of supernatant from cator (Bandelin SONOREX™, Germany). Biofilms were
non-turbid wells was plated on TSA, and the number of subsequently diluted and then placed on TSA. Mini-
colony-forming units (CFUs) was observed after 24 h of mum biofilm eradication concentrations at 50% and 90%
incubation at 37°C. MBC was defined as the minimum (MBEC50 and MBEC90) refer to the concentration of the
concentration of peptide at which no bacterial colony peptide at which established biofilm was eradicated by at
was observed on the plate [19]. least 50% and 90%, respectively [19, 20].

Hemolysis activity Serum stability

The effect of peptides on human red blood cells (hRBCs) The stability of BiF2_5K7K in the presence of human
was assessed by observing the release of hemoglobin serum was assessed by evaluating the MIC values, as
upon treated with peptide [16]. Briefly, the hRBCs were previously described [24]. Human serum was subjected
obtained from healthy volunteers and subsequently to heat inactivation at 56°C for 30 min. Bacterial suspen-
diluted in 10 mM PBS to a concentration of 2% hRBCs. sions were then adjusted to a concentration of 106 CFU/
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 5 of 19

mL in MHB supplemented with 25% and 50% human in MHB were exposed to BiF2_5K7K at MIC and/or
serum. These suspensions were mixed with two-fold seri- MBC for 24 h at 37°C. The bacterial suspensions were
ally diluted concentrations of BiF2_5K7K (ranging from obtained at specific time point (0, 2, 4, 6, 8 and 24 h), and
0.98 to 250 µg/mL) and incubated at 37°C for 24 h. MICs 10-fold serially diluted with PBS and then plated on TSA.
were subsequently determined as described above. The Untreated bacterial suspension was served as a control.
study protocol was ethically approved by the Thammasat After incubation, the number of CFUs was determined
University Ethics Committee (COA No. 066/2562). All by colony count.
volunteers provided written informed consent.
Confocal laser scanning microscopy
In vivo toxicity assay An Amsterdam active attachment (AAA) model was used
The toxicity of peptide BiF2_5K7K against human lung to assess the effectiveness of BiF2_5K7K to inhibit bio-
fibroblast MRC-5 cell lines (ATCC CCL-171) was inves- film formation as previously described [22]. In a 24-well
tigated by MTT colorimetric assay [16]. Briefly, MRC-5 microplate, various concentrations of BiF2_5K7K were
cells were cultured in Minimum Essential Medium incubated overnight with 107 CFU/mL of S. epidermidis.
[MEM; supplemented with 10% heat-inactivated FBS and The plate was then covered with a sanitary stainless-steel
1% Penicillin-Streptomycin] in 5% CO2 at 37°C. Follow- lid containing double-glass coverslips. Following 24 h of
ing an overnight incubation, MRC-5 cells were diluted incubation at 37°C, the biofilms were washed, and then
to 10,000 cells/well and placed in 96-well microplates. 50 mM green Alexa-ConA (Sigma-Aldrich, USA) was
Subsequently, they were exposed to BiF2_5K7K at 0.98 applied for 30 min to stain the biofilms. After staining,
to 250 µg/mL. After incubation for 24 h, MTT solution biofilms were fixed for 3 h with 2.5% glutaraldehyde. Bio-
(0.4 mg/mL) was added to each well and incubated for film images were acquired using a confocal laser scan-
an additional 3 h under 5% CO2 at 37°C. The supernatant ning microscope (CLSM; Carl-Zeiss/LSM800, Zeiss,
was then removed and 100 µL DMSO added, and absor- Germany). The fluorescence intensity of each test was
bance was measured at 570 nm using a microplate reader. analysed using ZEN 2.1 (blue edition) software.
The effect of BiF2_5K7K against mature biofilm was
Circular dichroism spectroscopy analysis also investigated. In short, overnight cultures of S. epider-
The conformational change of BiF2_5K7K in a membrane midis were diluted to 107 CFU/mL with TSB and further
mimicking environment was determined by measuring incubated at 37°C for 24 h to form biofilms. Next, wells
CD spectra of the peptide at wavelengths 190 to 250 nm were rinsed with sterile water and mature biofilms was
with a scanning speed of 10 nm/min by a Jasco-815 spec- treated with BiF2_5K7K at concentration of MBEC50 and
tropolarimeter (Jasco, Japan). As previously described further incubated for 24 h. Subsequently, biofilms were
[25], the CD spectra were obtained using a quartz cell washed again, then stained with green Alexa-ConA and
with a 1 mm path length at a temperature of 25°C. At a LIVE/DEADTM BacLight™ Bacterial Viability Kit (Invi-
final concentration of 0.2 mg/mL, BiF2_5K7K was dis- trogen™, Thermo Fisher, USA) for 15 min. After stain-
solved in three distinct membrane-mimicking environ- ing, 2.5% glutaraldehyde was used to fixed biofilms, then
ments, 10 mM PBS, 30 mM sodium dodecyl sulfate (SDS; observed under CLSM as described above.
Sigma-Aldrich, Japan), and 50% (v/v) 2,2,2-trifluoroetha-
nol (TFE; Merck, USA). PBS served as the aqueous sur- Transcriptional analysis
rounding, while the SDS micelles mimic the negatively The expression level of S. epidermidis biofilm forma-
charged environment of bacterial membranes featuring tion gene was investigated using a real-time reverse
an external surface with negative charges and an inter- transcription-polymerase chain reaction [27, 28]. In
nal hydrophobic environment resembling phospholipid brief, total RNA of S. epidermidis biofilm, both untreated
chains. TFE was utilized to simulate the hydrophobic and treated with BiF2_5K7K (at MBIC50 for 24 h), was
compartment of microbial membranes as the fluori- extracted by a RNeasy PowerBiofilm Kit (Qiagen, USA)
nated environment exhibits limited interaction with the and immediately reverse transcribed to cDNA by iScript
molecule and induce intra-molecular interactions that reserve transcription supermix (Biolabs, UK). RNApro-
frequently result in induced structuring, such as the for- tect Bacteria Reagent (Qiagen, USA) was used to preserve
mation of an alpha-helix structure [26]. An average of RNA during isolation. Remaining DNA was removed by
three scans were performed for each condition. treatment with DnaseI (Biolabs, UK). Real-time RT-PCR
was done in duplicate using iTaq Universal SYBR Green
Time-killing analysis Supermix (Bio-Rad, USA) with 5 ng of cDNA and 10 mM
The time-dependent bactericidal activity of BiF2_5K7K of forward and reverse primers [29, 30] (Table S1) using
against planktonic S. epidermidis was determined as pre- a CFX96 Real-Time PCR Detection System (Bio-Rad,
viously described [23]. In brief, mid-log phase bacteria USA). The cycling condition was started with an initial
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 6 of 19

denaturation at 95°C for 2 min, followed by 50 amplifi- segments by vortexing for 10 s, sonicating for 10 min, and
cation cycles consisting of 95°C for 30 s, 56°C for 1 min, vortexing again for 10 s. Cell suspensions were diluted
and 72°C for 1 min. Subsequently, a melting curve was and subsequently plated on TSA. The planktonic cells in
generated from 65°C to 95°C at intervals of 1 s at the end wells were evaluated by ten-fold serially diluting collected
of each run. To analyse the relative quantitation of gene supernatants and then plated on agar. After incubations,
expression, 2− ΔΔCt method was used, and 16S ribosomal the CFUs were counted.
subunit (rRNA) was served as the reference or house-
keeping gene. Statistical analysis
The experiment was performed at least three times. The
Inhibitory activity of BiF2_5K7K on segments of silicone results were displayed as the mean ± standard deviation
catheters (SD). One-way ANOVA or Student’s t-test were used
The capacity of BiF2_5K7K to prevent S. epidermi- to analyse the data via GraphPad Prism (version 9.0).
dis biofilm formation on a silicone catheter was deter- *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, and
mined using CV staining and colony count as previously ****p-value < 0.0001 were considered significant.
described [31, 32] with some modifications (Fig. 1). CV
staining was used to evaluate the development of bio- Results
film on BiF2_5K7K-coated catheters and the surround- Novel antibiofilm peptides were designed and
ing area, while the viability of bacteria and the number of characterized
planktonic cells adherent to the catheter was assessed by Through a template-based design strategy, a 7-amino
colony count. Briefly, a sterile silicone catheter (Norta®, acid cationic BiF peptide was designed from the con-
Malaysia) was sectioned into 50 mm segments which served, 18 α-helical sequence of antibiofilm peptides
were then coated with BiF2_5K7K at concentrations of derived from diverse species (including frogs, bovines,
250, 500, 1,000 or 2,000 mg/mL for 1 h by submerging sheep, fish, chickens, snakes, insects, and humans (Fig.
the catheter segments, followed by overnight air-drying. S1). Hydrophobicity, amphipathicity and net charge were
The pre-coated catheters were subsequently placed in a reported as the key parameters to designing novel pep-
24-well microplate containing S. epidermidis suspension tides with enhanced activity while reducing toxicity [14].
( ∼ 107 CFU/mL in TSB) and incubated at 37°C. Follow- Several studies have highlighted the significant influ-
ing 24 h of biofilm formation, supernatant was collected ence of amphipathicity, often referred as peptide helic-
for the colony count assay, and the wells were rinsed ity, on antibiofilm activity, as it directly affects the mode
thrice with 0.85% NaCl. of action [33]. Meanwhile, the antimicrobial activity of
To quantitate biofilm prevention, catheter segments peptides is predominantly impacted by their charge and
were stained for 15 min with a 0.1% CV solution, then hydrophobicity [25]. As shown in Table 1, in silico analy-
rinsed with 0.85% NaCl and resolved in 95% ethanol. sis of the parent peptide, BiF, revealed a positive charge
Biofilm formation on catheter segments was measured of +1 and high percentage (86%) of hydrophobic residues.
by a microplate reader at absorbance of 570 nm. Wells To increase the overall positive charge and the associ-
were also stained with the CV solution, as previously ated antibiofilm activity, BiF was subsequently modified
described. by hybridization at C-terminal segment with lipid bind-
To evaluate the viability of sessile and planktonic S. ing motif, KILRR, from NaD1 peptide [17]. The newly
epidermidis bacterial cells were removed from catheter obtained peptide, BiF2, with the net charge of +4 and

Fig. 1 Workflow of peptide coating on catheters

Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 7 of 19

67% hydrophobicity, was predicted to be an α-helical inhibitory activity against S. epidermidis biofilms, served
amphipathic structure with perfect helical wheel projec- as a positive control to assess biofilm inhibition [34]. The
tion in which the hydrophobic residue was located on MBIC50 and MBIC90 of vancomycin were both 3.91 µg/
one side and the charged residue on the other (Fig. 2). mL.
Subsequently, the hydrophobic leucine residue on the
polar face at positions 5 and 7 of BiF2 were substituted Modified peptide derivatives showed improved efficacy
with lysine (L5K, L7K) to obtain two hybrid derivatives, and safety
BiF2_5K and BiF2_5K7K. These derivatives retained The antibacterial activity of BiF and its derivatives against
amphipathic α-helical structures with increased overall planktonic biofilm-forming bacteria were determined.
positive charge to +5 and +6, and reduced hydrophobic When tested against target bacteria, the parent peptide
residues to 58% and 50%, respectively (Table 1; Fig. 2). BiF showed no antibacterial activity, while the modified
These peptide derivatives were chemically synthesized, peptides (with one and two lysine substitutions, BiF2_7K
and their molecular weights (MWs) were confirmed by and BiF2_5K7K) had broad-spectrum antibacterial activ-
electrospray ionization-mass spectrometry (ESI-MS). ity against biofilm-forming bacteria at concentrations
The correlation between the calculated and measured ranging from 7.81 to 250 µg/mL (Table 3). Moreover,
MW for each peptide indicated accurate synthesis of the these modified peptides also demonstrated lower hemo-
peptides [25]. lytic activity than did the parent peptide as shown in
Fig. 4. At a concentration of 31.25 µg/mL, more than 20%
BiF and its derivatives inhibited formation of S. epidermidis of hRBCs were lysed by BiF, and the lysis reached 100%
biofilm at a concentration of 250 µg/mL. In contrast, BiF2_7K
According to the result of inherent biofilm formation at the maximum concentration tested (250 µg/mL) dis-
capacity by CV staining assay, S. epidermidis ATCC played 60% hemolytic activity. Remarkably, BiF2_5K7K
35984 was classified as a strong biofilm-producing strain, displayed minimal hemolytic effect, measuring less than
as evidenced by its OD570 being four times higher than 10%, even at its maximum concentration. In contrast,
the ODc (data not shown). The ability of BiF and its the membrane-lytic melittin peptide, derived from bee
derivatives to prevent biofilm formed by S. epidermidis venom, used in hemolytic and antibacterial experiments
was further investigated. The concentrations of peptide as a positive control, exhibited potent antibacterial activ-
which inhibited 50% and 90% of biofilm formation were ity within the range of 3.91 to 15.63 µg/mL. Furthermore,
defined as MBIC50 and MBIC90, respectively. As shown in at 3.91 µg/mL, melittin completely lysed hRBCs.
Fig. 3, all peptide derivatives displayed a dose-dependent The therapeutic index (TI) is widely used for evaluating
inhibition of biofilm development. The parent peptide, a peptide’s efficacy and safety [35]. It is the ratio between
BiF, exhibited low biofilm inhibitory activity. However, the minimum hemolytic concentration (MHC, the con-
the lipid binding motif-conjugated peptide (BiF2) effec- centration causing 10% hemolysis) and the geometric
tively prevented the formation of biofilm with MBIC50 mean (GM) value of the MIC [15]. Greater TI values indi-
and MBIC90 values of 125 and 250 µg/mL, respec- cate that the peptide is more selective and specific for
tively (Table 2). The modified peptides, BiF2_7K and bacterial-like membranes than those of mammals [25].
BiF2_5K7K, exhibited higher biofilm inhibitory activity As shown in Table 3, BiF and BiF2, as well as the posi-
with MBIC50 values of 3.91 and 7.81 µg/mL, respectively, tive control melittin, exhibited very narrow therapeutic
and MBIC90 values of 7.81 and 31.25 µg/mL, respectively. windows (TI less than 0.2). While, both lysine-substi-
Vancomycin, a glycopeptide antibiotic known for its tuted peptides (BiF2_7K and BiF2_5K7K) had higher TIs

Table 1 The amino acid sequences of the peptide derivatives and their corresponding physicochemical characteristics
Peptide Sequence aaa Theoretical MWb Measured MWc Net charge <H> d <µH>e Pho%f
BiF FLVKLIL 7 845.14 844.58 +1 - - 86%
BiF2 FLVKLILKILRR 12 1512.00 1511.04 +4 0.784 0.603 67%
BiF2_7K FLVKLIKKILRR 12 1527.01 1526.05 +5 0.560 0.716 58%
BiF2_5K7K FLVKKIKKILRR 12 1542.02 1541.07 +6 0.336 0.679 50%
a
aa, number of amino acids
b
Theoretical MW, molecular weight calculated by https://aps.unmc.edu/prediction
c
Measured MW, molecular weight (g/mol) determined by mass spectroscopy (MS)
d
<H>, hydrophobicity and
e
<µH>, hydrophobic moment obtained from the website: http://heliquest.ipmc.cnrs.fr/
f
Pho%, the percentage of hydrophobic residues
-, No results were obtained
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 8 of 19

Fig. 2 The helical wheel projections of peptides A BiF2, B BiF2_7K and C BiF2_5K7K. Positively charged and hydrophobic residues are shown in blue and
yellow, respectively. The 3D structures are shown of D BiF2, E BiF2_7K and F BiF2_5K7K. Blue and green colors highlight positively charged and hydropho-
bic residues, respectively. The numbers represent the position of amino acid residues

against all tested biofilm-forming bacteria than did the BiF2_5K7K was still able to prevent the formation of bio-
two parent peptides, and the TI of BiF2_5K7K was higher film by S. epidermidis.
than that of BiF2_7K.
Based on its high inhibitory levels of biofilm-formation BiF2_5K7K showed no toxicity to lung fibroblasts
and low hemolytic activity, BiF2_5K7K was shown to be Since BiF2_5K7K demonstrated low hemolytic effect
a promising candidate peptide against biofilm-forming against hRBCs, its toxicity on the MRC-5 human embry-
bacteria. This assessment includes biofilm-forming S. onal lung fibroblast cell line was assessed through a MTT
epidermidis, widely known as one of the most preva- viability assay. In Fig. 5, BiF2_5K7K was not toxic (cell
lent causes of biofilm-related, medical device infections viability > 99%) toward the MRC-5 cell line even at the
[3]. At subinhibitory concentrations (<15.63 µg/mL), maximum concentration tested (250 µg/mL). In contrast,
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 9 of 19

Fig. 3 Inhibition of peptide derivatives against S. epidermidis biofilm compared to that of vancomycin. Data are presented as mean ± SD of at least three
independent experiments. Significance was determined by one-way ANOVA (**p-value < 0.01, ****p-value < 0.0001)

Table 2 The activities of peptide derivatives against S. dose-dependent cytotoxic effect.

epidermidis biofilm
Peptide MBIC50a (µg/mL) MBIC90b (µg/mL) BiF2_5K7K exhibited time- and concentration-dependent
BiF 125 >250 antibacterial activity and maintained its efficacy in human
BiF2 125 250 serum
BiF2_7K 3.91 7.81
The antibacterial activity of BiF2_5K7K against S. epi-
BiF2_5K7K 7.81 31.25
a b
dermidis was carried out with BiF2_5K7K at both MIC
MBIC50 and MBIC90, are defined as the minimum concentration of peptide
that can inhibit biofilm formation more than 50% and 90%, respectively and MBC concentrations (Fig. 6). In comparison to
the untreated control, both MIC and MBC concentra-
tions of BiF2_5K7K significantly decreased the num-
melittin, the positive control peptide, exhibited a strong ber of S. epidermidis (reaching approximately 103 CFU/

Fig. 4 The hemolytic activity of peptide derivatives on hRBCs. Tests were done in triplicate and reported as mean ± SD. Statistical significance was evalu-
ated using one-way ANOVA (*p-value < 0.05)
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 10 of 19

Table 3 The antibacterial activities (MIC and MBC values) of peptide derivatives against both gram-positive and gram-negative
bacteria
Bacterial strains MIC (MBC) (µg/mL)
Strain BiF BiF2 BiF2_7K BiF2_5K7K Melittin
Gram-positive bacteria
Staphylococcus epidermidis ATCC 35984a >250 31.25 7.81 15.63 15.63
(>250) (62.5) (7.81) (31.25) (15.63)
Staphylococcus aureus ATCC >250 62.5 15.63 >250 3.91
25923a (>250) (62.5) (15.63) (>250) (3.91)
Bacillus cereus ATCC 11778a >250 15.63 7.81 250 3.91
(>250) (15.63) (7.81) (250) (3.91)
Listeria monocytogenes 10403s >250 15.63 7.81 31.25 3.91
(>250) (31.25) (7.81) (62.5) (3.91)
Gram-negative bacteria
Pseudomonas aeruginosa ATCC 27853a >250 15.63 15.63 62.5 15.63
(>250) (31.25) (15.63) (125) (15.63)
Salmonella Typhimurium ATCC 13311a >250 62.5 7.81 31.25 3.91
(>250) (62.5) (15.63) (62.5) (3.91)
Escherichia coli ATCC 25922a >250 31.25 31.25 250 31.25
(>250) (31.25) (31.25) (250) (31.25)
Acinetobacter baumanii MT strainb >250 62.5 7.81 31.25 7.81
(>250) (62.5) (7.81) (31.25) (7.81)
MHCc (µg/mL) 15.63 1.95 15.63 >250 <0.98
GM +d >250 31.25 9.77 199.22 6.84
TI +e 0.03 0.06 1.60 2.51 0.07
GM-f >250 42.97 15.63 93.75 14.65
TI-g 0.03 0.05 1 5.33 0.03
a
ATCC, the American Type Culture Collection
b
MT strain, bacterial strain provided from Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University
c
MHC, minimum hemolytic concentration, indicated the peptide concentration causing 10% hemolysis of hRBCs. When no 10% hemolysis was detected at 250 µg/
mL, a value of 500 µg/mL was used to calculate the therapeutic index. When no 10% hemolysis was observed at a concentration of 0.98 µg/mL, a value of 0.49 µg/
mL was utilized for calculating the therapeutic index
d
GM + and f GM-, the geometric mean of MIC values from all gram-positive and -negative bacterial strains, respectively
e
TI + and g TI-, therapeutic index is the ratio of the MHC to the geometric mean of MICs from all gram-positive and -negative bacterial strains, respectively

Fig. 5 The cytotoxicity of BiF2_5K7K and melittin toward MRC-5 human embryonal lung fibroblast cell line. Experiments were performed in triplicate and
data are reported as mean ± SD. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, and ****p-value < 0.0001 indicate significant differences
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 11 of 19

Fig. 6 The time-dependent bactericidal activity of BiF2_5K7K at both MIC and MBC against S. epidermidis was evaluated at 0, 2, 4, 6, 8, and 24 h. Experi-
ments were conducted in triplicate, and results are presented as the mean ± SD. Statistical significance was denoted by *p-value < 0.05, **p-value < 0.01,
***p-value < 0.001, and ****p-value < 0.0001, indicating significant differences in the data

Fig. 7 Circular dichroism spectra of BiF2_5K7K in 10 mM PBS (blue), 50% TFE solution (black) and 30 mM SDS (red)

mL) within 4 h. At MBC, BiF2_5K7K completely killed mM PBS), the spectra of the peptide revealed a negative
S. epidermidis (more than 99% killing) within 6 h (while peak near 200 nm, indicating an unordered or random
regrowth was observed at MIC after 8 h). In the presence coil structure.
of 25% and 50% human serum, BiF2_5K7K exhibited no
change of antibacterial activity against S. epidermidis BiF2_5K7K inhibited S. epidermidis biofilm formation by
(MIC = 15.63 µg/mL), suggesting that BiF2_5K7K exhibits killing activity and altering biofilm gene expression
tolerable stability when exposed to human serum. The viability of S. epidermidis cells embedded in a bio-
film and exposed to various concentrations of BiF2_5K7K
BiF2_5K7K displayed α-helical structure in membrane- demonstrated a concentration-dependent bactericidal
mimicking environment activity, as shown in Fig. 8A. It was observed that at a
The secondary structure of BiF2_5K7K was examined concentration that inhibits bacterial growth, or MIC
by CD spectroscopy in various environments simulating (15.63 µg/mL), S. epidermidis biofilm cells remained
the bacterial membranes (Fig. 7). In 30 mM SDS and 50% viable. This result indicated a significant difference in
TFE solutions, BiF2_5K7K adopts a stable amphipathic the response between planktonic and biofilm cells to the
α-helical structure as shown by negative peaks at approx- peptide treatment. At higher concentrations (>15.63 µg/
imately 208 and 222 nm. In an aqueous environment (10 mL), there was no growth of biofilm cells, suggesting that
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 12 of 19

the peptide had a bactericidal effect operating through with the majority of bacteria staining red and their dis-
a mechanism similar to its action on planktonic cells. tribution uneven as compared to control (Fig. 9C). The
These results aligned with the biofilm inhibitory activity fluorescence intensity ratio (live to dead cells) demon-
shown by the crystal violet staining (Fig. 3), that at higher strated that there was a significant difference between
concentrations, the peptide exerted a killing effect which mature biofilm treated with BiF2_5K7K and untreated
prevented biofilm formation. However, at subinhibitory (p-value < 0.01) (Fig. 9D). Based on these findings, it
concentrations, crystal violet staining showed that the appeared that BiF2_5K7K was capable of damaging
biofilm inhibitory activity of BiF2_5K7K allowed the bio- mature S. epidermidis biofilm.
film cells to remain viable.
The impact of BiF2_5K7K at a subinhibitory concen- BiF2_5K7K prevented biofilm formation on silicone
tration [MBIC50 (7.81 µg/mL)] on biofilm formation was catheter
further evaluated by visualizing biofilm matrix labelled The inhibitory activity of BiF2_5K7K against S. epidermi-
with Alexa Fluor® 488. As shown in Fig. 8B, the reduction dis biofilm formation on a silicone catheter was evaluated
in S. epidermidis biofilm after treatment with 7.81 µg/ as a model for medical devices. A silicone catheter was
mL BiF2_5K7K was evident through the decrease in the coated with BiF2_5K7K by immersion in peptide solu-
green fluorescence emitted by Alexa Fluor® 488, compa- tion at concentrations of 250, 500, 1,000 or 2,000 µg/
rable to that of the untreated sample. These findings were mL, and the amount of released peptide from the cath-
further validated by assessing the fluorescence intensity eter was measured by fluorescamine protein assay. There
(Fig. 8C). For the S. epidermidis biofilm treated with were approximately 1.5, 4, 14, and 32 µg/mL of peptide
BiF2_5K7K at MBIC50, this was found to be significantly released, respectively (data not shown). Within 6 h,
lower than that of the untreated control (p-value < 0.001). coated catheters with BiF2_5K7K were able to limit the
The overall results indicated that BiF2_5K7K at MBIC50 adherence of S. epidermidis to the catheter surface, par-
did not inhibit bacterial growth but did reduce biofilm ticularly those coated with BiF2_5K7K at 2,000 µg/mL
development by S. epidermidis. (Fig. 10A). Coating resulted in a significant reduction
The effect of BiF2_5K7K on polysaccharide intercel- in the number of sessile bacteria (from 107 to 105 CFU/
lular adhesin (PIA)-producing genes (icaA, icaD, icaB, mL). Peptide released from the coated catheters signifi-
icaC, and icaR) and agr quorum-sensing genes (agrD, cantly inhibited the growth of planktonic cells (Fig. 10A).
agrB, agrC, and agrA) is shown in Fig. 8D and E, respec- Interestingly, the inhibition of biofilm development on
tively. Treatment with BiK2_5K7K at MBIC50 signifi- the BiF2_5K7K-coated catheter and surrounding area
cantly upregulated icaA, icaD, and icaB genes expression, was also observed (Fig. 10B), indicating the capability of
with the relative expression level greater than 1 at 24 h BiF2_5K7K not only directly inhibit biofilm formation on
(Fig. 8D); no alteration was noticed in levels of icaC or the catheter but also to diffuse from the coated catheter
icaR genes. In contrast, expression of the quorum- and inhibit biofilm formation within the surrounding
sensing genes agrD and agrB was reduced after 24 h of environment (wells).
BiF2_5K7K treatment of S. epidermidis biofilm compared Nevertheless, after 24 h, the attachment of sessile bac-
to that of the untreated control (Fig. 8E). However, nei- teria to the catheter surface and the growth of planktonic
ther agrC nor agrA gene expression changed at all over bacteria could only be inhibited on catheters coated with
this time period. 2,000 µg/mL of BiF2_5K7K (Fig. 10C). At all other con-
centrations, this effect was not observed. Even though the
BiF2_5K7K destroyed preformed S. epidermidis biofilm other BiF2_5K7K-coated catheters were unable to inhibit
BiF2_5K7K revealed a concentration-dependent eradica- attachment and growth of bacteria, they substantially
tion activity on established biofilm of S. epidermidis (Fig. inhibited the development of biofilm both on the cath-
S2). More than 90% of mature biofilm was eradicated eter itself and in the surrounding area (Fig. 10D). These
after 24 h of peptide treatment at a dosage of 125 µg/ findings were supportive of the capability of BiF2_5K7K
mL (MBEC90), while the MBEC50 was 31.25 µg/mL. The to prevent S. epidermidis biofilm development on cath-
destruction capability of BiF2_5K7K on mature biofilm eters. In this experiment, vancomycin was served as the
was confirmed by confocal imaging analysis. After treat- antibiotic control (Fig. S3).
ment with BiF2_5K7K at MBEC50, a significant decrease
in the S. epidermidis biofilm matrix was revealed by its Discussion
lower fluorescence intensity (Fig. 9A and B). In parallel, S. Medical devices play a crucial role in prolonging and
epidermidis biofilm cells viability after exposure to pep- enhancing quality of life, notably among the elderly
tide was determined by live/dead staining. Established S. [36]. During and after implantation, medical devices
epidermidis biofilm treated with BiF2_5K7K at MBEC50 can become contaminated with normal flora like S. epi-
exhibited a reduction in green fluorescence intensity, dermidis as well as other pathogenic organisms, causing
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 13 of 19

Fig. 8 Biofilm-formation inhibitory activity of BiF2_5K7K on S. epidermidis biofilm. A Biofilm cell viability after treatment with 0.98–250 µg/mL of BiF2_5K7K.
B Confocal imaging of S. epidermidis biofilm treated with BiF2_5K7K at MBIC50. C The fluorescence intensity ratio of Alexa Flour® 488 stained biofilms. D
and E The relative gene expression of S. epidermidis polysaccharide production and quorum-sensing genes, respectively. The data presented represent
the mean ± SD of at least three independent experiments. Statistical significance is denoted by *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, and
****p-value < 0.0001, indicating the presence of significant differences in the results
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 14 of 19

Fig. 9 Biofilm eradication activity of BiF2_5K7K on S. epidermidis biofilm. A Confocal imaging of preformed S. epidermidis biofilms after treatment with
BiF2_5K7K at MBEC50. B The fluorescence intensity ratio of Alexa Flour® 488 stained biofilms. C Live/dead results of S. epidermidis biofilms after treatment
with BiF2_5K7K at MBEC50. Live cells stained with SYTO 9 (green) while dead cells stained with propidium iodide (red). D The fluorescence intensity ratios
of live/dead cells analysed by ZEN 2.1. The presented data represents the mean ± SD derived from a at least three independent experiments. Statistical
significance is indicated by *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, and ****p-value < 0.0001, highlighting the presence of notable differences
in the findings

chronic infections, device failure and high mortality rates of action compared to conventional antibiotics [11, 12].
[5, 10]. The adherence of biofilm-forming microbes (such Some antibiofilm peptides incorporated onto medi-
as S. epidermidis) to medical devices makes this problem cal device surfaces have been shown to reduce initial
even worse [37], since biofilms are surrounded by an EPS attachment and, thus, biofilm development [10, 38, 39].
(composed predominantly of polysaccharide, extracellu- Moreover, antibiofilm peptides can be combined with
lar DNA (eDNA) and protein) which serves as a physi- antibiotics or other antibiofilm agents that act with dif-
cal barrier, protecting the bacteria within the biofilm ferent mechanisms and/or target-distinct biofilm fea-
from host defences and antimicrobial agents [10]. Conse- tures, and so improve overall antibiofilm activity [10, 16].
quently, the development of novel agents for treatment of By the sequence alignment of α-helical peptides with
biofilm-associated infections, especially medical device- antibiofilm activity, a consensus amphipathic sequence
related infections, is much needed. (FLVKLIL) rich in hydrophobic residues were retrieved
Rationally designed peptides have become a promising and served as a core antibiofilm peptide, BiF, for further
strategy for both inhibiting formation of biofilms and/ modification. Peptide hybridization provides an alterna-
or eradicating established biofilms. This is due to their tive method for promoting the activity and specificity of
potent and rapid antibiofilm and antimicrobial activ- peptides while minimizing their toxicity. In recent years,
ity against both planktonic and biofilm-embedded cells reports of combinations of peptides and motifs with
[10]. Additionally, they have a low likelihood of induc- specific functions have increased [40]. The hybrid pep-
ing bacterial resistance due to their distinct mechanisms tide R-FV-I16 was derived from RI16, an antimicrobial
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 15 of 19

Fig. 10 Antibiofilm activity of BiF2_5K7K on silicone catheter. A and C Bactericidal effect of BiF2_5K7K-coated catheters against sessile and planktonic S.
epidermidis after 6 and 24 h of incubation, respectively. B and D Biofilm inhibitory activity on BiF2_5K7K-coated catheter and surrounding area, respec-
tively. The experiments were done at least in triplicate; data are represented as the mean ± SD. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, and
****p-value < 0.0001 indicate significant differences

peptide, and combined with the antibiofilm motif FV7. with a lipid binding site motif not only enhanced antibio-
This hybrid peptide has potent antimicrobial and antib- film activity but also antibacterial activity. This enhance-
iofilm activities against both gram-positive and -negative ment is likely due to increased interaction between the
pathogenic bacteria, as well as reduced hemolytic activity lipid binding site of the peptides and the lipids present
and cytotoxicity [15]. We therefore introduced a highly in bacterial cell membranes [41]. Compared to the par-
positively charged lipid binding site motif (KILRR motif ent peptide, this incorporation did not lead to significant
from the NaD1 peptide) into the sequence of BiF at the differences in toxicity against human red blood cells. This
C-terminus to obtain a novel hybrid peptide (BiF2) with observation suggested that the decrease in hydrophobic-
higher positive charge and lower hydrophobicity. A pre- ity was not substantial enough to significantly enhance
vious study demonstrated that NaD1, an antifungal plant cell selectivity.
defensin, binds specifically to phospholipids, particularly A study by Von Borowski and colleagues revealed
phosphatidylinositol 4,5-bisphosphate (PIP2), via the K4 that lysine (K) and phenylalanine (F) are the most
residue and KILRR motif [17]. The incorporation of BiF found amino acids found in antibiofilm peptides [42].
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 16 of 19

Furthermore, their substitution with cationic amino acid naturally occurring proteins, showed slightly changes
residues, such as lysine or arginine (R), can increase the when exposed to human plasma [47].
positive charge of these peptides and enhance antimicro- BiF2_5K7K effectively inhibited S. epidermidis biofilm
bial activity [43, 44]. Subsequently, antibiofilm activity as development by more than 50% at MBIC50 (7.81 µg/mL).
one mechanism of action of these peptides was enhanced This inhibition was further confirmed by CLSM images,
along with their antimicrobial activity. There is evidence which demonstrated a reduction of the biofilm matrix.
that membrane disruption is the primary mechanism Moreover, at higher concentrations, BiF2_5K7K exerted
underlying their antibiofilm action [43]. Two hydropho- its biofilm-inhibiting effect by eradicating bacteria before
bic amino acids at positions 5 and 7 of BiF2 were substi- biofilm formation, indicating the biofilm inhibition is
tuted with positively charged lysines (L5K, L7K) in order achieved through its bactericidal activity. Therefore, to
to increase the positive charge and induce an amphipa- achieve complete inhibition (greater than 90% inhibition
thic structure of peptide characterized by a hydrophobic or MBIC90), a significantly higher concentration was nec-
surface on one side and a hydrophilic surface consisting essary. This finding suggested that at subinhibitory con-
of positively charged amino acid residues on the other. centration, BiF2_5K7K may have employed an alternative
Lysine was chosen over arginine because of its high heli- mechanism to combat biofilms rather than relying only
cal propensity and lower tendency to cytotoxicity [44]. on bactericidal actions [43].
As a result, BiF2_7K and BiF2_5K7K were obtained and It has been observed that there is no consistent cor-
both showed remarkable antibiofilm and antibacterial relation between antibacterial and antibiofilm activities.
activity. Strikingly, their effect toward human red blood While certain peptides demonstrate antibiofilm effects
cells was significantly decreased in comparison to the without affecting planktonic bacteria [10, 13], others
parent BiF2 peptide. These hybrid peptides and their possess the capacities for both antibacterial activity and
derivatives exhibited broad-spectrum activity against inhibition and/or elimination of biofilms [48]. BiF2_5K7K
both gram-positive and -negative bacteria. In contrast, is a peptide exhibiting dual functionality, encompass-
the parent peptide (BiF) displayed negligible antibacterial ing both antibacterial and antibiofilm activities. LL-37,
activity. The novel designed peptide BiF2_5K7K, which a well-known peptide with antibacterial and antibiofilm
integrates the parent sequence with a lipid binding site activities, displays broad-spectrum antibacterial activity.
motif and substitutes the amino acid at positions 5 and 7 Moreover, it has the capability to inhibit P. aeruginosa
with lysine, demonstrated higher antibiofilm and antibac- biofilm development through the promotion of twitching
terial activities while mitigating toxicity when compared motilities, downregulation of genes essential responsible
to that of parent peptide. This enhancement is attributed for biofilm formation, and the modulation of two key
to the optimization of the hydrophobicity, amphipathic- quorum-sensing systems [49]. The results of this study
ity, and net charge of the parent peptide. corroborated previous observations, indicating that the
The TI value serves as a parameter in assessing the interference with genes responsible for S. epidermidis
efficacy and safety of these peptides for clinical applica- biofilm formation might serve as a central mechanism
tions [45]. Among the peptide derivatives, BiF2_5K7K underlying the biofilm-inhibiting property of BiF2_5K7K.
exhibited the highest TI values against both gram-pos- The primary component within the S. epidermidis bio-
itive and -negative bacteria compared to others, even film matrix is an extracellular polysaccharide referred
though BiF2_7K demonstrated greater antibiofilm and to as polysaccharide intercellular adhesin (PIA) or poly-
antibacterial activities. The TI value of peptides is influ- N-acetylglucosamine (PNAG), which is a product of the
enced by their hemolytic effects, and thus helps ensure intracellular adhesion (ica) operon that composes icaA,
their safety towards mammalian cells and guide their icaD, icaB, icaC, and icaR (regulator of the ica locus)
development [16]. In order to evaluate its potential for genes [50]. The icaA and icaD genes encode transmem-
further use, a study of the cytotoxicity of BiF2_5K7K brane protein N-acetyl-glucosamine oligomers. The
against MCR-5 cells was conducted. The results indicated longer oligomer chains are transported across the cyto-
that BiF2_5K7K was not toxic towards mammalian cells. plasmic membrane by the IcaC protein and deacetylated
The stability of peptides in serum is crucial for evaluat- by the IcaB protein for cell surface attachment. The icaR
ing their effectiveness in environments resembling in gene has a regulatory role in repressing transcription at
vivo conditions [46]. When exposed to 25% and 50% the ica locus [51, 52]. Following exposure to BiF2_5K7K,
human serum, BiF2_5K7K maintained its antimicrobial gene expression within the ica operon includes upregula-
activity, indicating its potential applicability in biologi- tion of the icaA, icaD, and icaB genes during the adhe-
cal settings. The staphylocidal activity of BP2, a small sion and biofilm formation stages. These observations
synthetic amphipathic peptide designed based on the suggested that the altered ica operon expression con-
lipopolysaccharide-binding domains found in various tributes to the inhibition of biofilm development by
BiF2_5K7K at subinhibitory concentrations.
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 17 of 19

The human β-defensin 3 (hBD‑3) peptide exhibits anti- Amphipathicity, often referred to as peptide helicity
bacterial activity against S. aureus and effectively inhibits [55], plays a pivotal role in determining how peptides
its biofilm formation. This biofilm inhibition is attrib- interact with bacterial membranes. Peptide helicity has
uted to the impact of hBD-3 on genes related to biofilm been demonstrated by examining the secondary struc-
development. At a subinhibitory concentration (8 µg/ tural changes induced by electrostatic interactions
mL), hBD-3 demonstrates the ability to inhibit biofilm between the peptide and the bacterial membrane [45]. In
development by modulating the expression of the ica this study, BiF2_5K7K exhibited an amphipathic α-helical
operon. A substantial upregulation of the ica operon was conformation. The circular dichroism spectra further
observed when compared to the control [53]. Moreover, validated the secondary structure of BiF2_5K7K under a
the increased biofilm gene expression of S. epidermidis membrane-mimicking condition. While the structure of
may be a protective mechanism against the presence of BiF2_5K7K remained disordered in aqueous solution, an
the peptide, thereby preserving cell viability. This find- α-helical amphipathic structure was observed under dif-
ing is supported by previous studies which show that in fering membrane-mimicking environments, suggesting
response to environmental stress, treatment with rifam- its antibiofilm and antimicrobial activities.
picin, clindamycin, gentamicin, and vancomycin alone or Eliminating mature biofilms poses a considerable chal-
in combination affect the expression of the icaA and rsbU lenge in biofilm treatment, as extracellular matrix may
genes [28, 53]. constitute up to 90% of an established biofilm, encasing
During the development of biofilm, the quorum-sens- the bacteria and delaying or preventing antibiofilm agent
ing system is the regulatory system that strongly impacts penetration into the biofilm structure [10, 16]. Inter-
biofilm development [8]. One of the most studied quo- estingly, previous research has highlighted the unique
rum-sensing systems in S. epidermidis is the accessory potential of peptides in combating biofilms. Such efficacy
gene regulator (agr) system. The agr quorum-sensing can be attributed to the amphipathic properties and flex-
system is comprised of two different transcription units, ible molecular structures of the peptides, enabling them
identified as RNAII and RNAIII. The RNAII locus con- to readily penetrate biofilm structures. This enhanced
sists of four genes: agrB, agrD, agrC, and agrA. The pres- ability to infiltrate the extracellular matrix is a crucial fac-
ence of BiF2_5K7K may have reduced the levels of agrD tor contributing to their efficacy in preventing and dis-
and agrB gene expression, causing interference with the rupting biofilms. In this study, BiF2_5K7K demonstrated
agr quorum-sensing system. Downregulation of the agrD the ability to eradicate mature S. epidermidis biofilms
and agrB genes leads to inhibition of RNAIII, which is and this was likely due to their amphipathic properties
the main effector molecule of the agr quorum-sensing and flexible structures, allowing free biofilm penetration
system. The BCp12 peptide, a novel peptide derived [56]. However, higher concentrations of the peptide were
from buffalo casein hydrolysate, demonstrates a compa- needed for this eradication compared to that required to
rable capability to BiF2_5K7K by effectively preventing prevent initial development. This suggested that function
the formation of S. aureus biofilm at both the MIC and of the peptide may be limited by the presence of extracel-
sub-MIC concentrations. This inhibition occurs through lular matrix.
the downregulation of agrA, agrB, agrC, and psmβ gene Urinary catheters represent one of the commonly used
expressions, resulting in interference of the agr quorum- devices in healthcare settings. However, their long-term
sensing system [54]. Based on our findings, BiF2_5K7K use is limited by the risk of catheter-associated urinary
inhibited S. epidermidis biofilm formation by inducing tract infections (CAUTI) which may cause up to 23%
environmental stress, and thereby modifying the expres- of infections in intensive care units (ICUs) and 40% of
sion of polysaccharide production and downregulat- hospital-related infections [57, 58]. Most (70–80%) of
ing the agr quorum-sensing system. The ica operon in these infections occur owing to bacterial contamination
S. epidermidis plays an important role in device-related either before or during insertion [59], and often develop
infection. Therefore, altering this operon could be a cru- highly drug-resistant biofilm. Coating catheters with
cial step in combating bacterial biofilm infections [52]. antibiofilm peptides represents an attractive approach to
The downregulation of genes in the agr quorum-sensing limiting this problem. These peptides have been demon-
system implied a disruption in bacterial communication strated to have potent antibiofilm and broad-spectrum
mechanisms, suggested a potential to inhibit biofilm for- antibacterial activities while causing minimal toxicity and
mation, which is a crucial step in combating bacterial inducing resistance. As a preliminary test, BiF2_5K7K
biofilm infection [8]. By interfering with these systems, was immobilized onto a catheter via physical adsorp-
BiF2_5K7K could prevent the ability of bacteria to estab- tion. Approximately 1% (0.4 to 1.6%) of BiF2_5K7K was
lish biofilm and enhance the efficacy of conventional distributed along the catheter, indicating that physical
antibiotic treatments when used in combination. adsorption may not be the optimal method for coat-
ing catheters. Despite this, the results indicated that
Wongchai et al. Annals of Clinical Microbiology and Antimicrobials (2024) 23:44 Page 18 of 19

Acknowledgements
a BiF2_5K7K-coated catheter can inhibit S. epidermi- This work was supported by the Thailand Science Research and Innovation
dis biofilm formation on the catheter surface as well as Fundamental Fund fiscal year 2023, National Research Council of Thailand
in the surrounding area, though it cannot prevent the (NRCT) (13/2563), and Thammasat University Research Unit in Antimicrobial
Agents and Application. Miss Mathira Wongchai was supported by a Ph.D.
attachment of sessile and planktonic bacteria. Previous scholarship from Thammasat University. We would like to thank Dr. Arthur
research suggested that peptide coatings typically exhibit Brown, Faculty of Medical Technology, Mahidol University, Thailand, for
less antimicrobial activity than their soluble forms, providing language assistance.

maybe attributed to the non-specific nature of immobi- Author contributions

lization techniques [59]. The biofilm inhibitory activity of MW: Conceptualization, Methodology, Investigation, Data curation,
the BiF2_5K7K-coated catheter was found to be depen- Writing-Original draft., SW: Data curation, Methodology., SK: Data curation,
Methodology., SR: Resource, Data analysis., RA: Conceptualization,
dent on the concentration of the coating. Higher concen- Visualization, Investigation, Supervision, Data curation, Writing-Reviewing and
trations exhibited greater efficacy in inhibiting biofilm Editing, Funding Acquisition.
formation on both the catheter surface and its surround-
Data availability
ing areas. However, no correlation was observed between All of the data produced throughout this research are contained within this
the ability to prevent bacterial attachment on the coated article and/or its supplementary information file.
catheter and that in the surrounding area. These findings
may be attributed to the concentration retained within or Declarations
released from the catheter. To enhance the activity of the
Ethics approval and consent to precipitate
coated catheter through adsorption techniques, very high This research was approved by the Thammasat University Ethics Committee
concentrations may be required, or alternative methods (COA No. 066/2562) and all volunteers provided written informed consent.
for coating peptides such as cysteine-immobilization
Consent for publication
and polydopamine coating onto the catheter surface may Written informed consent for publication was obtained.
need to be explored. In a study by Mishra et al., Lasio-
III was chemically modified with a cysteine residue and Competing interests
The authors declare no competing interests.
subsequently immobilized on silicon surfaces. The result-
ing cysLasio-III-immobilized catheters demonstrated Author details
1
antimicrobial activity against E. coli and Enterococcus Graduate Program in Biomedical Sciences, Faculty of Allied Health
Sciences, Thammasat University, Pathum Thani, Thailand
faecalis, along with antibiofilm properties, showing a 2
School of Dentistry, King Mongkut’s Institute of Technology Ladkrabang,
reduction of E. faecalis and E. coli biofilms by 60% and Bangkok, Thailand
3
40%, respectively [60]. Similarly, in another study by Lim Department of Microbiology, Faculty of Medicine, Khon Kaen University,
Khon Kaen, Thailand
et al., the CWR11 tethered onto catheter surfaces using 4
Functional Proteomics Technology Laboratory, National Center
the polydopamine coating technique exhibited no visible for Genetic Engineering and Biotechnology, National Science and
colonies when incubated with E. coli [61]. In addition, Technology Development Agency, Pathum Thani, Thailand

combining antibiofilm peptide with antimicrobial pep-

Received: 11 January 2024 / Accepted: 28 April 2024
tides, conventional antibiotics, or compounds with spe-
cific functions such as Dnase (to dissolve biofilm matrix)
[62], QS inhibitor [10], and disodium ethylenediamine-
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