Synergistic and antibiofilm properties of ocellatin peptides against multidrug-resistant Pseudomonas aeruginosa.

TL;DR: Ocellatin-PT3 may be promising as a template for the development of novel antimicrobial peptides against P. aeruginosa, and was capable of preventing the proliferation of 48-h mature biofilms at concentrations ranging from 4 to 8× the MIC.

Abstract: Aim To test ocellatin peptides (ocellatins-PT2-PT6) for antibacterial and antibiofilm activities and synergy with antibiotics against Pseudomonas aeruginosa. Materials & methods Normal- and checkerboard-broth microdilution methods were used. Biofilm studies included microtiter plate-based assays and microscopic analysis by confocal laser scanning microscopy and atomic force microscopy. Results Ocellatins were more active against multidrug-resistant isolates of P. aeruginosa than against susceptible strains. Ocellatin-PT3 showed synergy with ciprofloxacin and ceftazidime against multidrug-resistant isolates and was capable of preventing the proliferation of 48-h mature biofilms at concentrations ranging from 4 to 8× the MIC. Treated biofilms had low viability and were slightly more disaggregated. Conclusion Ocellatin-PT3 may be promising as a template for the development of novel antimicrobial peptides against P. aeruginosa. [Formula: see text].

Summary

Introduction

• To test ocellatin peptides (ocellatins-PT2-PT6) for antibacterial and antibiofilm activities and synergy with antibiotics against Pseudomonas aeruginosa.
• Normal- and checkerboard-broth microdilution methods were used, also known as Materials & methods.
• Indeed, biofilms of P aeruginosa are one of the bottlenecks in the treatment of such infections .
• In the last few years, many AMPs have been reported as promising novel antimicrobial drugs due to their main mechanisms of action, which include disrupting membranes, interfering with metabolism, and targeting cytoplasmic components [13].

Materials & methods

• Antimicrobial agents & ocellatin peptides Standard laboratory powders of ceftazidime and ciproB.oxacin hydrochloride were purchased from SigmaAldrich (MO, USA).
• All the antibiotic discs used were from Oxoid (Basingstoke, England).
• The peptides were syn thesized using the Merrifield solid phase synthesis techniques on a 24 channel multiplex Symphony® peptide synthesizer (Gyros Protein Technologies, Inc, AZ, USA) and were assembled using 0(6chlorobenzotriazollyl).
• Bacterial strains & growth conditions P. aeruginosa ATCC 27853, P. aeruginosa PAOl and a susceptible clinical isolate, PA007, as well as MDR clinical isolates of P. aeruginosa (PalSA2, Pa4SA2 and PA006) were used in this study.

MIC & MBC determination

• The minimum inhibitory concentration (MIC) values of the five ocellatins, ceftazidime and ciproB.oxacin against P. aeruginosa isolates were determined by the broth microdilution method, following the recommendations contained in the Clinical and Laboratory Standards Institute (CLSI) guidelines [24], with the exception that MH broth was used instead of cationadjusted MH broth.
• The MIC was defined as the lowest concentration that completely inhibited the growth of bacteria as detected by the naked eye.
• The minimum bactericidal concentration (MBC) was determined by spreading 10 µl on MH agar from the wells corresponding to/ and above the MIC showing no visible growth, with further incubation for 24 h at 37°C; the lowest concentration at which no bacterial growth occurred on MH plates was defined as the MBC.
• These experiments were performed in three independent experiments.

Synergy testing

• The discdiffusion method on agar was used as a screening test to assess the combined effect between ocellatins and antibiotics.
• MDR P. aeruginosa isolates from fresh cultures in MH were suspended in buffered peptone water in order to reach a turbidity equal to a 0.
• The plates were incubated overnight at 37°C.
• Potential synergism was inferred when the zone of inhibition caused by the antibiotic discs impregnated with ocellatins was greater than the inhibition zone produced by the antibiotic discs or peptideimpregnated blank discs alone.
• BrieB.y, FIC of drug A (FIC A) = MIC of drug A in combination/MIC of drug A alone, and FIC of drug B (FIC B) = MIC of drug B in combination/MIC of drug B alone.

Biofilm inhibition assay

• Given the promising antibacterial activity of ocellatinPT3 against P. aeruginosa isolates, its ability to inhibit the biofilm formation was assessed.
• Bacterial suspensions without ocellatinPT3 were used as controls.
• Briefly, biofilms were allowed to form for 48 h in 96well microtiter plates, then the planktonic phase were discarded and the biofilms were rinsed twice and further treated with different concentrations of ocellatinPT3 ranging from the MIC value up to 12x MIC.
• All experiments were performed at room temperature, and each chamber slides was used for no longer than 10 min.

Statistical analysis

• The biofilm inhibition and treatment assays as well as the biofilm metabolic activity assay were carried out in two independent experiments, being each experiment performed in triplicate.
• The results of the biofilm formation were expressed as mean values ± standard deviation.
• The statistical significance of differences between controls and experimental groups was evaluated using Student's t-test.
• Probability values (p) of < 0.05 were considered statistically significant.

Results

• Antibacterial activity of ocellatin peptides against P. aeruginosa MIC values of ocellatins were initially determined against a P. aeruginosa ATCC 27853 and an MDR isolate, Pa4 SA2.
• Interestingly, the MIC and MBC values were lower against Pa4SA2 than against the reference strain.
• The screening for potential synergy between ocellatins and antibiotics against MDR P. aeruginosa isolates revealed that the combinations of ocellatinPT3/ceftazidime and ocellatinPT3/ciproBoxacin increased (by 34 mm) the zones of inhibition in comparison to the zones caused by each compound alone.
• Those combinations were further tested using a checkerboard method.
• Only the synergies between ocellatinPT3/ceftazidime and ocellatinPT3/ciproBoxacin were confirmed (FIC index :::;0.5; Table 3).

Antibiofilm activities of ocellatin-PT3

• The ability of ocellatinPT3 to inhibit the biofilm formation by Pa4SA2 and PA006 isolates was examined (Figure 1).
• Additionally, AFM images (Figure 4) of the biofilms formed and treated in the same conditions as for CLSM analysis reflect also a disaggregating effect on the biofilm caused by the ocellatinPT3 as well as a direct effect on bacterial cells, which became more wrinkled and seem hollow.
• Others have also described a particular AMP, T9W, to have strong and specific activity against P. aeruginosa and low or no activity against other Gram negative and Grampositive bacteria [33].
• Hence, the CD results suggest an interaction between ocellatinPT3 and LPS isolated from P. aeruginosa that may represent the begin ning of the mechanism of bactericidal action.

Conclusion & future perspective

• Over the last years, AMPs have gained increasing attention as potential novel antimicrob ial drugs alternatives for combating infections caused by antibioticresistant bacteria and/ or associated to biofilms.
• Ocellatin-PT3 may be explored for the design and development of novel antimicrobial peptides.
• The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Figures

Figure 1. Biomass quantification of biofilms of Pseudomonas aeruginosa Pa4-Sa2 and PAOOG. Biofilms were formed in the presence of different concentrations of ocellatin-PT3 (ranging from 1/ B x MIC to MIC). Differences between the experimental group and the control were statistically significant for a *p < 0.05. MIC: Minimum inhibitory concentration.

Figure 2. Metabolic activity reduction of Pseudomonas aeruginosa biofilms after treatment with ocellatin-PT3. 48-h preformed biofilms of Pa1-SA2, Pa4-SA2 and PA006 were treated for 24 h with a concentration of ocellatin-PT3 corresponding to the respective minimum biofilm inhibitory concentration . The metabolic activity was assessed through the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Differences were statistically significant in relation to the control for a ***p < 0.001 .

Figure 3. Confocal laser scanning microscopy qualitative evaluation of Pseudomonas aeruginosa biofilms not treated (controls) and treated with ocellatin-PT3. 48-h preformed biofilms of Pa1-SA2 (A), Pa4-SA2 (B) and PA006 (C) were treated for 24 h with a concentration of ocellatin-PT3 corresponding to the respective minimum biofilm inhibitory concentration. All biofilms, after live/dead staining, show viable (green fluorescence) and dead (red fluorescence) cells. Percentages of viable and dead cells are also shown; the values are the means ± standard deviation obtained from the counts of six representative images taken from each biofilm that was visualized .

Figure 4. Atomic force microscopy images of Pseudomonas aeruginosa biofilms not treated (A) and treated (B) with ocellatin-PT3. 48-h preformed biofilms of Pa1-SA2, Pa4-SA2 and PA006 were treated for 24 h with a concentration of ocellatin-PT3 corresponding to the respective minimum biofilm inhibitory concentration.

Full text

Synergistic a nd a ntibiofilm properties of ocel latin
peptides against
multidrug-resista nt
Pseudomonas
aeruginosa
Lucinda
J
Bessa*·
1
,
Peter Eaton
1
,
Anderson Dematei
2
•
5
,
Alexandra Placido
3
,
Nuno Vale
4
,
Paula Gomes
1
,
Cristina Delerue-Matos
3
,
Jose Roberto SA Leite
1
•
5
& Paula Gameiro
1
1
LAQV/REQUIMTE, Departamento de Quimica e Bioquimica, Faculdade de Ciencias da Universidade do Porto, 4169-007 Porto,
Portugal
2
Ciencias Quimicas e das Biomoleculas, Centro de lnvestigaao em Saude e Ambiente, Escola Superior de Saude, Institute
Politecnico do Porto, 4200-072 Porto, Portugal
3
LAQV/REQUIMTE, Departamento de Engenharia Quimica, Institute Superior de Engenharia do Institute Politecnico do Porto,
4200-072 Porto, Portugal
4
UCIBIO/ REQUIMTE, Laborat6rio de Farmacologia, Departamento de Ciencias do Medicamento, Faculdade de Farmacia da
Universidade do Porto, 4050-313 Porto, Portugal
5
Area de Morfologia, Faculdade de Medicina, Universidade de Brasilia, UnB, Brasilia, 70910-900 Brasil
* Author for correspondence: lucinda.bessa@fc.up.pt
Aim:
To test ocellatin peptides (ocellatins-PT2-PT6) for antibacterial and antibiofilm activities and synergy
with antibiotics against Pseudomonas aeruginosa.
Materials & methods:
Normal- and checkerboard-broth
microdilution methods were used. Biofilm studies included microtiter plate-based assays and microscopic
analysis by confocal laser scanning microscopy and atomic force microscopy.
Results:
Ocellatins were more
active against multidrug-resistant isolates of
P.
aeruginosa than against susceptible strains. Ocellatin-PT3
showed synergy with ciprofloxacin and ceftazidime against multidrug-resistant isolates and was capable
of preventing the proliferation of 48-h mature biofilms at concentrations ranging from 4 to 8x the MIC.
Treated biofilms had low viability and were slightly more disaggregated.
Conclusion:
Ocellatin-PT3 may
be promising as a template for the development of novel antimicrobial peptides against
P.
aeruginosa.
Ocellatin peptides
(ocellatin-PT3)
Synergy with antibiotics
Antibiofilm activity
P. aeruginosa biofilm Ocellatin-PT3-treated
e MIC alone
D MIC in combination

Keywords: AMPs • antibiofilm activity • antimicrobial peptides • multidrug-resistant Pseudomonas
aeruginosa •
ocellatin peptides • ocellatin-PT3 • synergy
Antimicrobial resistance (AMR) is one of the greatest threats to health today [I,2]
.
It
can affect anyone, at any
age, in any country.
It
is of consensus that the threat of drug resistance can only be tackled with the right set of
actions, including the development of novel treatment options and alternative antimicrobial therapies [3]. Gram
negative pathogens are of particular concern since they are becoming resistant to nearly all the antibiotic drug
options available, in fact, infections by pandrugresistant Gramnegative bacilli are rising [4]. The most worrisome
Gramnegative infections are commonly caused by Enterobacteriaceae (mostly Klebsiella pneumoniae), Pseudomonas
aeruginosa and Acinetobacter spp. [5].
P aeruginosa
is a ubiquitous microorganism that commonly causes hospitalacquired infections, including
pneumonia, bloodstream and urinary tract infections and it is well known for chronically colonizing the respiratory
tract of patients with cystic fibrosis, causing severe intermittent exacerbation of the condition
[6].
P aeruginosa
infections are particularly difficult to control because of its high level of intrinsic resistance to antibiotics due
primarily to a combination of the impermeable outer membrane and a number of broadspectrum efflux pumps
[7].
Some strains of
P aeruginosa
have been found to be multidrugresistant (MDR), with resistance to nearly all
antibiotics, including aminoglycosides, cephalosporins, Buoroquinolones and carbapenems
[5].
Moreover, this
Gramnegative bacterium is capable of forming structured aggregates known as biofilms, which definitely contribute
to the increase of both pathogenicity and resistance to antibiotic treatment
[8,9].
Biofilm formation by
P aeruginosa
is the result of a complex adaptation process driven by genetic variation and the qualitative composition of the
polysaccharide content in the biofilm matrix is highly dependent on phenotypic features including the ability to
synthesize high amounts of alginate (mucoid strains) or Psl/ Pel (nonmucoid strains)
[IO].
Indeed, biofilms of
P
aeruginosa
are one of the bottlenecks in the treatment of such infections . Therefore, there is an urgent need of novel
antimicrobial agents and treatment strategies able to effectively counteract planktonic as well as biofilm modes of
growth.
Antimicrobial peptides (AMPs) historically have been called defensive molecules, and are believed to be the first
line of the innate immune response system against viruses, bacteria and fungi
[I I,12].
Natural AMPs can be found
in every organism from prokaryotes to eukaryotes (protozoan, fungi, plants, insects and animals)
[I I].
In the last few years, many AMPs have been reported as promising novel antimicrobial drugs due to their
main mechanisms of action, which include disrupting membranes, interfering with metabolism, and targeting
cytoplasmic components
[13].
Additionally, AMPs are increasingly being considered as novel agents against biofilms
by inhibiting the biofilm formation or eradicating established biofilms
[14,15].
Another approach to overcome the problem of MDR bacteria is by combining different drugs. The combination
of AMPs with commercially available antibiotics have also been explored as a potential alternative for combating
drugresistant infections caused by several microorganisms [1618].
In this study, five peptides

ocellatinPT2PT6

previously isolated from the skin secretion of the frog
Leptodactylus pustulatus [19] were tested for antimicrobial activity and synergistic effects with antibiotics against
P aeruginosa. The peptides herein studied were part of a set of eight new AMPs, called ocellatins, which had
been isolated from the crude skin secretion of L. pustulatus, identified and tested for antimicrobial activity against
Escherichia coli, Staphylococcus aureus, K pneumoniae and Salmonella choleraesuis strains by Marani et al. [19].
Moreover, those ocellatins were reported to present little or no hemolytic activity against human erythrocytes and
no cytotoxicity against murine fibroblasts.
Recently, several frog skinderived AMPs have been largely reported to show antibacterial properties
[2023].
There
have also been reports of antibiofilm activity from some such molecules
[15].
Therefore, the skin secretions from
many species of anurans are a rich source of peptides with antimicrobial activities that should be explored for
further research and development of novel therapeutic agents.

.
Table
Amino acid sequence and molecular weight of ocellatin-PT2-PT6.
Peptides
Sequence
MW Ref.
Ocellatin-PT2
GVFDllKDAGKQLVAHATGKIAEK vt 2609.0
[19]
Ocellatin-PT3
GVIDllKGAGKDLIAHAIGKLAEKV 1 2530.0
[19]
Ocellatin-PT4
GVFDllKGAGKQLIAHAMGKIAEKV 1 2595.1
[19]
Ocellatin-PT5 GVFDllKDAGRQLVAHAMGKIAEKV 1 2667.1 [19]
Ocellatin-PT6
GVFDllKGAGKQLIAHAMEKIAEKVGLNKDGN
3365.9 [19]
t c-terminus-amidated peptide.
MW: Molecular weight.
Materials & methods
Antimicrobial agents & ocellatin peptides
Standard laboratory powders of ceftazidime and ciproB.oxacin hydrochloride were purchased from SigmaAldrich
(MO, USA). All the antibiotic discs used were from Oxoid (Basingstoke, England). The five ocellatin peptides
(ocellatinPT2PT6), whose amino acid sequences are shown in Table 1, were manually synthesized, purified
and quantified through slightly different protocols than those previously described
[19].
The peptides were syn
thesized using the Merrifield solid phase synthesis techniques on a 24 channel multiplex Symphony® peptide
synthesizer (Gyros Protein Technologies, Inc, AZ, USA) and were assembled using 0(6chlorobenzotriazollyl)
N,N,N',N'tetramethyluronium hexaB.uorophosphate and N,Ndiisopropylethylaminecoupling conditions. The
full and detailed protocol of synthesis and purification is provided as supplementary material. All peptides were
dissolved in MilliQ water to obtain stock solutions of 10 mg/ ml.
Bacterial strains & growth conditions
P.
aeruginosa
ATCC 27853,
P.
aeruginosa
PAOl and a susceptible clinical isolate, PA007, as well as MDR clinical
isolates of
P.
aeruginosa
(PalSA2, Pa4SA2 and PA006) were used in this study. The AMR profile of MDR isolates
is shown in Supplementary Table 1. These bacteria were grown on MuellerHinton (MH) agar (Liofilchem srl,
Roseto degli Abruzzi [Te], Italy) from stock cultures. MH plates were incubated at 37°C prior to obtain fresh
cultures for each
in vitro
bioassay.
MIC & MBC determination
The minimum inhibitory concentration (MIC) values of the five ocellatins, ceftazidime and ciproB.oxacin against P.
aeruginosa isolates were determined by the broth microdilution method, following the recommendations contained
in the Clinical and Laboratory Standards Institute (CLSI) guidelines [24], with the exception that MH broth was
used instead of cationadjusted MH broth. The MIC was defined as the lowest concentration that completely
inhibited the growth of bacteria as detected by the naked eye. The minimum bactericidal concentration (MBC) was
determined by spreading 10 µl on MH agar from the wells corresponding to/ and above the MIC showing no visible
growth, with further incubation for 24 h at 37°C; the lowest concentration at which no bacterial growth occurred
on MH plates was defined as the MBC. These experiments were performed in three independent experiments.
Synergy testing
The discdiffusion method on agar was used as a screening test to assess the combined effect between ocellatins
and antibiotics. MDR P. aeruginosa isolates from fresh cultures in MH were suspended in buffered peptone water
(Oxoid) in order to reach a turbidity equal to a 0. 5 McFarland standard and spread on MH agar plates. CiproB.oxacin
and ceftazidime discs were used as controls and were also impregnated with 15 µl of a 10mg/ ml solution of each
peptide. The plates were incubated overnight at 37°C. Potential synergism was inferred when the zone of inhibition
caused by the antibiotic discs impregnated with ocellatins was greater than the inhibition zone produced by the
antibiotic discs or peptideimpregnated blank discs alone.
Based on the results of the previous assay, potential synergism observed between ocellatins, particularly ocellatin
PT3 and ocellatinPT4, and antibiotics (ciproB.oxacin or ceftazidime), was then checked using a broth microdilution
checkerboard method and tested against Pa4SA2 and PalSa2 as previously described
[25].
Three independent
experiments were carried out. The fractional inhibitory concentrations (FIC) were calculated and interpreted as
stated by Gomes
et al.
[25].
BrieB.y, FIC of drug A (FIC A)
=
MIC of drug A in combination/MIC of drug A alone,
and FIC of drug B (FIC B)
=
MIC of drug B in combination/MIC of drug B alone. The FIC index (I;FIC) is the

sum of each FIC and is interpreted as follows: I;FIC
:S
0.5, synergy; 0.5
<
I;FIC
:S
4, indifference; 4
<
I;FIC,
antagonism.
Biofilm inhibition assay
Given the promising antibacterial activity of ocellatinPT3 against
P.
aeruginosa
isolates, its ability to inhibit the
biofilm formation was assessed. OcellatinPT3 at concentrations equal to MIC, l /2 x MIC, l/4 x MIC and
l/8 x
MIC was added to bacterial suspensions of 1 x 10
6
CFU/ml in tryptic soy broth. Bacterial suspensions
without
ocellatinPT3 were used as controls. Each suspension was dispensed into a 96well microtiter plate
(200 µI/well)
and incubated at 37°C for 48 h. After that time, biofilms were stained with 0.5% crystal violet for 5 min, rinsed with
water, air dried and eluted with acetic acid 33%
(v/ v).
The optical density was measured at
595 nm.
Biofilm treatment assay
The efficacy of ocellatinPT3 on established biofilm of P. aeruginosa was also assessed by obtaining the minimum
biofilm inhibitory concentration (MBIC). Briefly, biofilms were allowed to form for 48 h in 96well microtiter
plates,
then the planktonic phase were discarded and the biofilms were rinsed twice and further treated with different
concentrations
of ocellatinPT3 ranging from the MIC value up to 12x MIC. The optical density (OD)6oo was
immediately measured
and measured again after 24 h of incubation at 37°C. The MBIC was defined as the lowest
concentration of ocellatin
inhibiting the bacterial proliferation in the planktonic phase, confirmed by no increase
or :::;10% increase in the optical
density compared with the initial reading [26].
Evaluation of biofilm metabolic activity
48h biofilms of PalSA2, Pa4SA2 and PA006 formed as described above in 96well microtiter plates were
subsequently treated with ocellatinPT3 at a concentration equivalent to the respective MBIC. After 24 h of
incubation at 37°C, the bacterial metabolic activity of biofilms was quantified using the 3(4,5dimethylthiazol2 yl)
2,5diphenyltetrazolium bromide

MTT (0.5 mg/ml; SigmaAldrich) for 3 h at 37°C in the dark. Dimethyl sulfoxide
(DMSO) was used to extract the formazan dye product and then absorbance at 570 nm was measured.
Confocal laser scanning microscopy
For confocal laser scanning microscopy (CLSM) analysis, 48h biofilms of PalSA2, Pa4SA2 and PA006 were
formed in µDish (35 mm, high), ibidi Polymer Coverslips (ibidi GmbH, PlaneggMartinsried, Germany) from
starting inocula of 1 x 10
6
CFU
I
ml in tryptic soy broth. After 48 h, biofilms were rinsed twice with phosphate
buffered saline and treated with a concentration of ocellatinPT3 equal to the respective MBIC for 24 h. Biofilms
were
then rinsed and stained using the live/ dead staining BacLight bacterial viability kit (Molecular Probes, Thermo
Fisher
Scientific, MA, USA). Biofilms were examined by a widefield fluorescence microscope Zeiss Axiolmager
Zl equipped
with a PlanApochromat 63x /1.40 Oil DIC objective and a camera Axiocam MR ver.3.0 (Carl
Zeiss, Oberkochen,
Germany) and by a laser scanning confocal system Leica TCS SP5 II using a HC PL APO CS
63x /1.30 Glycerine 21°C
objective (Leica Microsystems, Wetzlar, Germany). All experiments were performed at room temperature, and each
chamber slides was used for no longer than 10 min.
As for the viability cell counts, the proportion oflive and dead cells was determined by counting six representative
images
taken from each biofilm visualized, using software Image analysis [27].
Atomic force microscopy imaging
48h biofilms of PalSA2, Pa4SA2 and PA006 were formed on a glass coverslip previously put inside 35mm
diameter polystyrene plates and treated with or without ocellatinPT3 as above described for CLSM. Biofilms formed on the
coverslips were rinsed with sterile phosphatebuffered saline and dried before atomic force microscopy (AFM)
imaging.
Samples were scanned with a TTAFM from AFMWorkshop in air in vibrating mode. A 50µm scanner
and 300 kHz
silicon cantilevers (ACT, AppNano, CA, USA) were used. Images were processed using Gwyddion
2.47 software.
Conformational analysis of ocellatin-PT3 by circular dichroism
The secondary structure content of ocellatinPT3 was assessed by circular dichroism (CD) spectroscopy in the far
UV,
using a Jasco J815 CD Spectropolarimeter QASCO) as previously reported [28]. Briefly, the measurements

Table 2. Minimum inhibitory concentration and minimum bactericidal concentration values (fig/ml) of ocellatins against
susceptible and multidrug-resistant
Pseudomonas
aeruginosa.
Peptides P. aeruginosa ATCC P. aeruginosa PAQ11 PA0071 (MIC [MBC]) Pa4-SA21 (MIC [MBC]) Pa1-SA21 (MIC [MBC]) PA0061 (MIC [MBC])
278531 (MIC [MBC]) (MIC [MBC])
Ocellatin-PT2 1024 (>1024) 512 (>1024) 1024 (>1024) 128 (256) 256 (512) 16 (32)
Ocellatin-PT3 >512 (-) 512 (>1024) 1024 (>1024) 64 (128) 128 (256) 16 (16)
Ocellatin-PT4 >512 (-) 512 (>1024) 1024 (>1024) 256 (256) 512 (1024) 16 (32)
Ocellatin-PT5 >512 (-) 512 (>1024) 1024 (>1024) 128 (256) 256 (512) 32 (64)
Ocellatin-PT6 1024 (>1024) 512 (>1024) 1024 (>1024) 128 (256) 256 (512) 32 (64)
!Susceptible.
1Multidrug-resistant.
MBC: Minimum bactericidal concentration; MIC: Minimum inhibitory concentration.
were carried out under a nitrogen gas Bow of 8 l/ h at 20°C. Spectra were obtained between 190 and 260 nm.
The lipopolysaccharides (LPS) from P. aeruginosa were obtained from SigmaAldrich. OcellatinPT3 was used in a
concentration of lOO µM and the LPS in concentrations of O, 0.50, 0.75 and 1.00% (p/ v) in MilliQ water. These
experiments were performed at 37°C and a scan speed of 50 nm/ min, a response time of 1 s and a bandwidth of
1 nm were used. The spectra were converted to molar ellipticity per residue as previously reported [28,29].
Statistical analysis
The biofilm inhibition and treatment assays as well as the biofilm metabolic activity assay were carried out in
two independent experiments, being each experiment performed in triplicate. The results of the biofilm formation
were expressed as mean values
±
standard deviation. The statistical significance of differences between controls
and experimental groups was evaluated using Student's
t-test.
Probability values (p) of < 0.05 were considered
statistically significant.
Results
Antibacterial activity of ocellatin peptides against P. aeruginosa
MIC values of ocellatins were initially determined against a P. aeruginosa ATCC 27853 and an MDR isolate, Pa4
SA2. Interestingly, the MIC and MBC values were lower against Pa4SA2 than against the reference strain.
Therefore, subsequently, we have determined the MIC of that ocellatin against other two nonMDR (susceptible)
and two MDR P. aeruginosa isolates (Table 2). OcellatinPT3 was the most active among the five peptides with
lower MIC and MBC values. As shown, the activity against the clinical isolate PA006 was particularly strong, the
highlight being the bactericidal activity of ocellatinPT3 at only 16 µg/ ml.
Synergy between ocellatins & antibiotics
The screening for potential synergy between ocellatins and antibiotics against MDR
P.
aeruginosa
isolates revealed
that the combinations of ocellatinPT3/ceftazidime and ocellatinPT3/ciproBoxacin increased (by 34 mm) the
zones of inhibition in comparison to the zones caused by each compound alone. Equally, the combinations
ocellatinPT4/ceftazidime and ocellatinPT4 also increased growth inhibition (by 23 mm) compared with single
components. Photos of the combined effect between ocellatins and antibiotics against Pa4SA2 can be seen in
Supplementary Figure 1. Those combinations were further tested using a checkerboard method. Only the synergies
between ocellatinPT3/ceftazidime and ocellatinPT3/ciproBoxacin were confirmed (FIC index :::;0.5; Table 3).
Antibiofilm activities of ocellatin-PT3
The ability of ocellatinPT3 to inhibit the biofilm formation by Pa4SA2 and PA006 isolates was examined
(Figure 1). In presence of concentrations equal to the MIC and l /2 x MIC, less biofilm biomass was quantified.
However, at lower subinhibitory concentrations more biofilm was formed compared with the control.
The MBIC of ocellatinPT3 against 48h established biofilms formed by the three MDR isolates are shown in
Table 4. The concentrations of ocellatinPT3 that could inhibit the proliferation of mature biofilms ranged from 4
to lO x the respective MIC values. The relative MBIC of ciproBoxacin was considerably higher than that recorded
for this peptide. For instance, when a 48h biofilm of Pa4SA2 was treated with a concentration 32 x MIC of

Frequently asked questions

Q1. What are the contributions in this paper?

Ocellatin-PT3 showed synergistic effects with ciprofloxacin and ceftazidime, which might hold promise for developing future combinatorial therapies after uncovering the mechanisms triggering such synergy this paper. 

Q2. What is the effect of ocellatin-PT3 on the biofilm?

Ocellatin-PT3 could inhibit the proliferation of established biofilms at concentrations from 4 to 10x the MIC, which was mostly due to a direct killing effect on the bacterial cells within the biofilm as shown by confocal laser scanning microscopy after live/dead staining . 

Q3. What is the reason for the higher activity of ocellatins against resistant strains?

The explanation for the higher activity of ocellatins against drugresistant isolates than against susceptible strains may be related to membrane permeability / impermeability. 

Q4. What is the mechanism behind the ocellatin peptides?

It is known thatpermeability mutations are responsib le for increasing the resistance to many classes of antimicrobials (lactams, Buoroquinolones, aminoglycosides) in P. aeruginosa [34], therefore, those mutations, present in the MDR isolates, may revert a natural impermeability to ocellatins, suggesting that the development of resistance to known antibiotics and to ocellatins may be mechanistically independent. 

Q5. What was the concentration of ocellatinPT3 in MilliQ water?

OcellatinPT3 was used in a concentration of lOO µM and the LPS in concentrations of O, 0.50, 0.75 and 1.00% (p/ v) in MilliQ water. 

Q6. What is the role of ocellatin PT3 in the development of novel antim?

OcellatinPT3 may be promising as a lead molecule for the design and development of novel AMPs withsignificant activity and selectivity against MDR P. aeruginosa biofilms. 

Q7. What is the mechanism of action of ocellatinPT3?

Among the five ocellatin peptides tested, ocellatinPT3 acted synergistically with ceftazidime and ciproBoxacin against MDR isolates. 

Q8. How much less activity did ocellatinPT3 have on biofilms?

The authors could also observe that biofilms treated with ocellatinPT3 in a concentration equal to the MBIC had a much lower metabolic activity (around 70% less activity in respect to controls). 

Q9. What is the MIC of the biofilms treated with ocellatinPT3?

AFM images (Figure 4) of the biofilms formed and treated in the same conditions as for CLSM analysis reflect also a disaggregating effect on the biofilm caused by the ocellatinPT3 as well as a direct effect on bacterial cells, which became more wrinkled and seem hollow. 

Q10. What is the effect of ocellatinPT3 on biofilms?

in order to explore the effects of ocellatinPT3 on the biofilms viability, CLSM was used in conjunction with the live/ deadstaining technique, revealing in fact a marked decrease in the viability of bacterial cells within the ocellatinPT3treated biofilms. 

Q11. What is the MIC of ocellatin against P. aeruginosa?

The screening for potential synergy between ocellatins and antibiotics against MDR P. aeruginosa isolates revealed that the combinations of ocellatinPT3/ceftazidime and ocellatinPT3/ciproBoxacin increased (by 34 mm) the zones of inhibition in comparison to the zones caused by each compound alone. 

Q12. What is the role of ocellatinPT3 in the biofilms?

In fact, the microscopic analyses showed that ocellatinPT3 could also affect the biofilms structure, by causing a slight disaggregation. 

Q13. What is the normal structure of the biofilm?

This is normal, since AFM only shows the upper surface of the biofilm, which for mature biofilms typically consist more of the polymeric matrix than individual cells. 

Q14. How did ocellatinPT3 inhibit the growth of mature biofilms?

the combinations ocellatinPT4/ceftazidime and ocellatinPT4 also increased growth inhibition (by 23 mm) compared with single components. 

Q15. What was the MIC of ocellatinPT3 in combination with antibiotics?

MIC values of ocellatins-PT3 and -PT4 in combination with antibiotics and respective fractional inhibitory concentration index values obtained from the checkerboard method. 

Q16. What was the biofilm inhibition and treatment assay?

The biofilm inhibition and treatment assays as well as the biofilm metabolic activity assay were carried out in two independent experiments, being each experiment performed in triplicate. 

Q17. How long did the biofilms remain in the dark?

After that time, biofilms were stained with 0.5% crystal violet for 5 min, rinsed with water, air dried and eluted with acetic acid 33% (v/ v). 

Q18. What is the role of AMPs in the treatment of bacterial infections?

Over the last years, AMPs have gained increasing attention as potential novel antimicrob ial drugs alternatives for combating infections caused by antibioticresistant bacteria and/ or associated to biofilms. 

Q19. What is the effect of ocellatin on biofilms?

The CLSM images (Figure 3) showed, for all three isolates, a clear effect of ocellatinPT3 in lowering the viability ofthe bacterial cells within the biofilm. 

Q20. What was the MIC of ocellatinPT3 against the clinical isolate?

As shown, the activity against the clinical isolate PA006 was particularly strong, the highlight being the bactericidal activity of ocellatinPT3 at only 16 µg/ ml. 

Q21. What is the main component of the outer membrane in Gramnegative bacteria?

This is then followed by cell internalization, presumab ly mediated by the bacterialLPS (i.e., the major component of the outer membrane in Gramnegative bacteria) [39].