Presentation given during Cost AMiCI meeting in Tallinn Nov 2017 by Maria Olivia Pereira, Assistant Professor University of Minho Department of Biological Engineering Braga, Portugal, Professor in Biomedical Engineering Principal Investigator in the Biofilm Group

1. COST-AMICI meeting
Tallinn
November 15-17, 2017
ANTIMICROBIAL PEPTIDES & THEIR
POTENTIAL TO COMBAT
ANTIMICROBIAL RESISTANCE
Maria Olivia Pereira
mopereira@deb.uminho.pt
OPINION ON THE USE OF ANTIMICROBIAL
COATINGS IN HEALTH-CARE SETTINGS

2. Context/Motivation
Maria Olivia Pereira
Assistant Professor
University of Minho
Department of Biological Engineering
Braga, Portugal
PhD in Biological and Chemical Engineering
Professor in Biomedical Engineering
Principal Investigator in the Biofilm Group (https://www.ceb.uminho.pt/People/Details)
Center of Biological Engineering (CEB) (https://www.ceb.uminho.pt)
Research Area: Health Biotechnology and Bioengineering
Research Group: BIOFILM (https://www.ceb.uminho.pt/biofilm)
Research interests:
• Investigation of the virulence and resistance phenomena of microorganisms towards antimicrobials
• Development of effective therapeutic strategies to fight biofilm- and devices-associated infections
based on innovative drugs (antimicrobial peptides, enzymes, natural products), drugs combinations
and in-situ drug release.
- Development of functionalized anti-adhesive and anti-biofilm surfaces;
- Design of nanoparticles-based therapies to drugs delivery;.

3. Widespread and unnecessary use and
misuse of antibiotics
Antimicrobial Resistance
Biofilm resistance
Nosocomial, biomaterial and chronic
related infections
• Organized, structured and adhered consortia of microorganisms
protected by a matrix
• Cause among 80 % of bacterial infections
• Main cause of dissemination of resistance in the nosocomial scenario
• Resistance to the immune system  chronic infections
BIOFILMS
+
Biofilm formation stages
3
Selection of MDR Pathogens
e.g. ESKAPE group
23 000 deaths/year USA
700 000 deaths/year worldwide
Enterococcus faecium
Staphylococcus aureus
Klebsiella pneumoniae
Acinetobacter baumannii
Pseudomonas aeruginosa
Enterobacter species
A N T I M I C R O B I A L R E S I S TA N C E

4. Combination strategies with
efficacy potentiation
New antimicrobials with new
modes of action
A N T I M I C R O B I A L R E S I S TA N C E
Widespread and unnecessary use and
misuse of antibiotics
Antimicrobial Resistance
Biofilm resistance
Nosocomial, biomaterial and chronic
related infections
+
• Antimicrobial peptides (AMP) • AMP combinations
• AMP-Traditional Antimicrobials combinations
• Antimicrobials immobilization
?

5. • Short-length peptide antibiotics (10 to 30 a.a.)
• Found throughout Nature
• Wide-spectrum
• Immune system of animals and plants
• Cationic and directed to the cell membrane (majority)
• Multiple mechanisms of action
• Low specificity of molecular target
• Support antimicrobial action (e.g. cytokine release, chemotaxis,
antigen presentation, angiogenesis and wound healing)
Publications related to AMP throughout time
Publications related to biofilms and AMP throughout time
A N T I M I C R O B I A L P E P T I D E S ( A M P )

6. • Increase the antimicrobial spectrum
• Prevent the emergence of resistance
• Lower individual agent dosage
o Reduce toxicity and side effects
• Provide synergistic activity
Accumulation of
Data
Combination studies
Bioinformatics
Approaches
Text-mining
Network
reconstruction
Network Exploration
Unveil new
combinations
Publications related to AMP combinations throughout time
AMP Combinations ?
A N T I M I C R O B I A L C O M B I N AT I O N S

7. i. Susceptibility profiles of P. aeruginosa and S. aureus (planktonic and biofilm cultures):
ii. Prophylactic and therapeutic approaches (prevention of biofilm growth vs eradication
of grown biofilms).
iii. Single- and double-species biofilms of P. aeruginosa and S. aureus.
iv. Cytotoxicity of the combinations against mammalian cells.
P. aeruginosa PAO1
S. aureus ATCC 25923
P. aeruginosa U147016-1
P. aeruginosa I92198-1 (MDR)
S. aureus GB 2/1
S. aureus PD95.2 (MRSA)
Reference
Clinical isolates (CI)
Colistin (CST)
Citropin 1.1
(CIT 1.1)
Tachyplesin I linear
analogue (TP-I-L)
Temporin A
(TEMP-A)
I N V I T R O S T U D I E SBiofilm-associatedinfections
AMP alone
AMP combinations

8. Planktonic Single AMP Susceptibility
I N V I T R O S T U D I E S
Biofilm-associatedinfections
Strain
CST TEMP-A CIT-1.1 TP-I-L
MIC MBC MIC MBC MIC MBC MIC MBC
P. aeruginosa
PAO1 2 2 >128 >128 128 128 8 8
CI 2 2 >128 >128 128 >128 8 8
CI MDR 2 4 >128 >128 128 >128 16 32
S. aureus
ATCC 25923 64 >128 8 16 16 16 4 8
CI 32 128 4 8 8 8 2 4
CI MRSA 32 >128 4 32 8 32 8 32
Values in mg/L
 Best outcomes with CST and TP-I-L
 S. aureus overall more susceptible, except for CST
 MDR/MRSA slightly less susceptible

9. Strain
CST +
TEMP-A CIT-1.1 TP-I-L
FIC FBC FIC FBC FIC FBC
P. aeruginosa
PAO1 S S S S S S
CI S S S S S Ad
CI MDR Ad S Ad ND Ad Ad
S. aureus
ATCC 25923 Ad Ad I Ad S S
CI Ad Ad Ad I Ad S
CI MRSA Ad ND Ad ND Ad S
CST range for synergy:
• 0.25 – 1 mg/L for P. aeruginosa
• 1 – 16 mg/L for S. aureus
 Almost all combinations were synergic for P. aeruginosa
 S. aureus overall less susceptible, except for CST+TP-1-L
 MDR/MRSA slightly less susceptible
ND – not detected in the range of concentrations tested.
Checkerboard Assay
Synergy S
Additiveness Ad
Indifference I
Antagonism A
Planktonic Combined AMP Susceptibility
I N V I T R O S T U D I E S
Biofilm-associatedinfections

10. Prophylactic Approach – Biofilm Growth Inhibition
e.g. S. aureus
Comparison with the action of the most active
individual AMP
Synergy (S) ≥ 2 log decrease
Additiveness (Ad) 1 ≤ log < 2 decrease
Indifference (I) < 1 log decrease
Antagonism (A) ≥ 2 log increase
Statistical differences / Biological significance
Synergy (S) combination action > sum of the isolated actions
Additiveness (Ad) combination action = sum of the isolated action
Indifference (I) combination action = action of the most active peptide alone
Antagonism (A) combination action < action of the most active peptide alone
I N V I T R O S T U D I E S
Single-species Biofilm Combined AMP Susceptibility
Biofilm-associatedinfections

11. Strain
CST +
TEMP-A CIT-1.1 TP-I-L
P. aeruginosa
PAO1 Ad / Ad (7.0) S / S (7.7) Ad / I (7.3)
CI S / S (5.7) S / S (6.3) S / S (6.1)
CI MDR S / Ad (4.9) I / I (3.2) Ad / Ad (6.2)
S. aureus
ATCC 25923 S / S (4.8) A / I (1.7) S / I (3.7)
CI I / I (3.5) S / Ad (3.3) S / Ad (5.8)
CI MRSA S / Ad (4.9) Ad / Ad (2.9) S / S (4.6)
Statistical / Biological significance
Prophylactic Approach – Biofilm Growth Inhibition
CST range for synergy:
• 4 – 8 mg/L for P. aeruginosa
• 16 – 32 mg/L for S. aureus
 Most combinations were synergic or additive
 P. aeruginosa CI more susceptible
 Some combinations reached total inhibition
In parenthesis ():
Highest average log
(CFU/cm2)
reduction achieved
compared to the
positive control
Synergy S
Additiveness Ad
Indifference I
Antagonism A
I N V I T R O S T U D I E S
Single-species Biofilm Combined AMP Susceptibility
Biofilm-associatedinfections

12. Statistical / biological significance; (highest average log (CFU/cm2) reduction achieved compared to the positive control), ≥2 log reductions are shown in bold
Strain
CST +
TEMP-A CIT-1.1 TP-I-L
2 h 6 h 2 h 6 h 2 h 6 h
PBS TSB PBS TSB PBS TSB PBS TSB PBS TSB PBS TSB
P. aeruginosa
PAO1
S / I
(1.1)
S / I
(0.5)
I / I
(1.0)
A / I
(1.0)
A / I
(1.0)
I / I
(0.6)
I / I
(0.6)
I / I
(0.5)
A / I
(1.0)
I / I
(0.8)
S / I
(2.1)
I / I
(0.7)
CI
S / Ad
(3.4)
I / I
(1.8)
S / S
(3.2)
Ad/ I
(2.3)
S / I
(2.9)
S / Ad
(3.6)
S / Ad
(3.9)
S / Ad
(3.9)
S / Ad
(3.5)
A / I
(1.5)
S / Ad
(2.8)
I / I
(2.2)
CI MDR
I / I
(0.2)
S / I
(0.4)
I / I
(0.6)
S / Ad
(1.4)
I / I
(0.4)
S / I
(0.5)
S / I
(1.5)
S / Ad
(2.0)
I / I
(0.0)
Ad/ I
(0.2)
A / I
(0.2)
I / I
(1.2)
S. aureus
ATCC 25923
Ad/ I
(1.7)
S / I
(1.7)
I / I
(2.7)
A / I
(1.4)
I / I
(0.5)
I / I
(1.3)
A / I
(0.6)
Ad/ I
(1.8)
I / I
(2.7)
Ad/ I
(2.3)
I / I
(3.6)
I / I
(1.3)
CI
S / S
(2.5)
A / I
(0.5)
I / I
(1.1)
A / I
(1.2)
I / I
(0.9)
I / I
(1.0)
A / I
(0.1)
I / I
(1.0)
I / I
(1.9)
S / I
(0.9)
I / I
(2.9)
S / I
(1.9)
CI MRSA
I / I
(1.4)
A / I
(1.1)
I / I
(1.1)
I / I
(1.1)
I / I
(0.3)
I / I
(0.4)
I / I
(0.1)
I / I
(0.0)
I / I
(1.1)
A / I
(0.4)
I / I
(0.8)
I / I
(0.3)
CST range for synergy:
• 32 mg/L for P. aeruginosa
• 64 mg/L for S. aureus
 More positive outcomes for P. aeruginosa CI
 2 h treatment slightly better for TEMP-A
 PBS treatment slightly better for all combinations
Therapeutic Approach – 24 h-old Biofilm Eradication
Synergy S
Additiveness Ad
Indifference I
Antagonism A
I N V I T R O S T U D I E S
Single-species Biofilm Combined AMP Susceptibility
Biofilm-associatedinfections

13. CFU Growth Media TSA PIA MSA
Treatment time 2 h 6 h 2 h 6 h 2 h 6 h
CST +
P. aeruginosa PAO1 + S. aureus ATCC 25923
TP-1-L
S / Ad
(3.8)
S / Ad
(3.4)
Ad / Ad
(3.6)
S / S
(4.6)
I / Ad
(1.6)
I / I
(2.1)
TEMP-A
I / I
(0.9)
Ad / I
(0.9)
I / I
(0.2)
Ad / I
(0.3)
I / I
(0.7)
Ad / I
(0.7)
CIT-1.1
A / I
(0.4)
Ad / I
(1.4)
A / I
(0.2)
Ad / I
(0.5)
A / I
(0.5)
A / I
(0.5)
P. aeruginosa CI + S. aureus CI
TP-1-L
I / Ad
(4.8)
Ad / Ad
(3.8)
Ad / Ad
(4.3)
I / I
(3.1)
I / I
(2.2)
I / I
(2.8)
TEMP-A
I / I
(1.6)
A / I
(2.1)
A / I
(1.8)
I / I
(2.5)
Ad / Ad
(1.2)
I / Ad
(1.5)
CIT-1.1
I / I
(2.0)
I / I
(2.7)
I / I
(1.9)
I / I
(2.3)
Ad / Ad
(1.2)
Ad / Ad
(2.2)
P. aeruginosa CI MDR + S. aureus CI MRSA
TP-1-L
I / I
(0.7)
S / I
(1.0)
Ad / Ad
(1.5)
Ad / Ad
(1.3)
S / S
(4.5)
Ad / Ad
(3.2)
TEMP-A
Ad / Ad
(0.7)
Ad / Ad
(1.3)
Ad / Ad
(1.1)
S / Ad
(1.8)
S / I
(0.4)
S / Ad
(1.8)
CIT-1.1
Ad / I
(0.9)
S / Ad
(2.7)
Ad / I
(1.3)
S / S
(3.8)
I / I
(0.0)
S / S
(2.5)
Statistical / biological significance; (highest average log (CFU/cm2) reduction achieved compared to the
positive control), ≥2 log reductions are shown in bold
CST range for synergy:
• 16– 128 mg/L
 Treatment time had little influence
 Better results for reference and CI
MDR/MRSA biofilms
 Outcomes were CFU media dependent
 Higher conc. compared with single-
species:
• S. aureus small colony variants
• S. aureus lysis by P. aeruginosa
Therapeutic Approach – 24 h-old Biofilm Eradication
Synergy S
Additiveness Ad
Indifference I
Antagonism A
I N V I T R O S T U D I E S
Double-species Biofilm Combined AMP Susceptibility
Biofilm-associatedinfections

14. AMP Cytotoxicity
CST
TP-1-L
TEMP-A
CIT-1.1
CST + TP-1-L
CST + TEMP-A
CST + CIT-1.1
AMP (mg/L) AMP (mg/L)
Fibroblasts BALB/3T3 after 24 h of contact with AMP
 Individual and combined AMP were non-toxic (cell viability above 70 %) at ≤ 32 mg/L (16 mg/L for CIT-1.1)
 CST was the least toxic AMP
 Although some toxicity was observed for higher conc., time of contact (24 h) was superior to AMP treatment (2 h,
6 h)
AMP alone AMP combined
• Consecutive treatments with lower AMP conc.
• Addition of matrix disrupting enzymes (MDE) to the AMP combinations
• Immobilization of AMP
I N V I T R O S T U D I E S
Biofilm-associatedinfections

15.  First public database on AMP combinations
(http://sing.ei.uvigo.es/antimicrobialCombination/)
3111 combinations
 Overall synergy or additiveness outcomes, even for MDR double-species biofilms
 Biofilm treatment more challenging than biofilm growth prevention
 Higher AMP concentrations required in the treatment of double-species vs single-species biofilms
 Non-toxic AMP combinations up to 32 mg/L after 24 h.c23 000 deaths/year USA
 c23 000 deaths/year USA
C O N C L U S I O N S
In Vitro studies
Biofilm-associatedinfections
 Surface immobilization to create antimicrobial coatings
 Nanooformulations (cubosomes) to release the drugs at site of infection

16. Context/Motivation
Antimicrobial coatings
Alves et al., Acta Biomaterialia, 44 (2016), 313-322
Polydimethylsiloxane (PDMS)
Antimicrobial
Non-toxicNo resistance
development
Anti-adhesive
Antimicrobial Lipopeptide (Palm)
Antimicrobial functionality
Enzyme (DNase I)
Anti-adhesive functionality
Multi-functional
coatings
F U N T I O N A L I Z AT I O N O F S U R F A C E S
Biomaterials-associatedinfections
pH 8.5
1.Dopamine 2. Mixture of DNase I and
Palm
OH
OH
OH
OH
1:3
Polydopamine film
(pDA)
Mussel-inspired Coating Strategy

17. Alves et al., Acta Biomaterialia, 44 (2016), 313-322
PDMS pDA MIX PDMS pDA MIX PDMS pDA MIX
0.0
0.5
1.0
1.5
2.0
Normalizedattachment[foldedoverPDMS]
Live
Dead
P. aeruginosaS. aureus P. aeruginosa
+
S. aureus
24 h
Artificial urine
F U N T I O N A L I Z AT I O N O F S U R F A C E S
Biomaterials-associatedinfections
Coating Strategy performance
P. aeruginosaS. aureus
PDMS pDA MIX PDMS pDA MIX
0.0
0.2
0.4
0.6
0.8
1.0
OD490nm
 Coatings exhibited strong antimicrobial and
anti-adhesive features
 Similar results for single and co-adhesion
 However, outcomes were growth media
dependent
• Efficiency decreases when a complex
media was used, specially for the Gram-
positive bacteria.
24 h
TSB

18. Context/Motivation
Role of host immunity
Resistance
Susceptibility to antibiotic
treatment
What’s the fate of bacteria that managed to adhere to these surfaces?
• Are they more susceptible or did they develop some resistant phenotype?
• Are they more prone to be cleared by the host immune system?
Polydimethylsiloxane (PDMS)
F AT E O F B A C T E R I A
Biomaterials-associatedinfections
?

19. Context/Motivation
Biofilm Susceptibility to Vancomycin Therapy
0,0
0,8
1,6
2,4
PDMS pDA pDA-MIX
A490nm
No treatment Treatment [MIC]
Metabolic activity of cells
XTT reduction assay (OD490 nm)
(***) p < 0.001, compared to No treatment
***
1x107 CFU/mL
24 h, 37 oC, 120 rpm + overnight vancomycin treatment
Staphylococcus aureus
A N T I B I O T I C S U S C E P T I B I L I T Y
Biomaterials-associatedinfections
 Cells that managed to adhere to PDMS were
able to grow into biofilms
 Vancomycin treatment had no effect on
biofilms formed on unmodified PDMS and on
PDS+pDA
 Vancomycin caused a significant reduction of
the metabolic activity of biofilms formed on
the functionalized PDMS
• Synergistic effect between those coatings
and antibiotic therapy

20. Context/Motivation
Potential of Immobilized Compounds to Induce Bacterial Resistance
S. aureus
3 or 4 days, @120 rpm, 37 oC
Cells detached
Sonication + vortex
1x107 CFU/mL
TSB
Experimental Design
30 days
R E S I S TA N C E D E V E L O P M E N T
Biomaterials-associatedinfections
Control surfaces
•PDMS
•pDA
Antimicrobial
surfaces
•MIX
•Vancomycin
(VANC)
MIC and MBC
Transcriptomic Analysis

21. Context/Motivation
MIC and MBC
Antimicrobial
MIC MBC
PDMS pDA MIX/VANC PDMS pDA MIX/VANC
Palm 32-128 32-128 32-128 64-128 64-128 64-128
Vancomycin 1-2 1-2 1-2 2-8 (16) 4-16 4-16 (32)
Antimicrobial
MIC MBC
PDMS pDA MIX/VANC PDMS pDA MIX/VANC
Palm 32-128 32-128 32-128 64-128 64-128 64-128
Vancomycin 1-2 1-2 1-2 2-8 (16) 4-16 4-16 (32)
Antimicrobial
MIC (µg/mL) MBC (µg/mL)
PDMS pDA MIX/VANC PDMS pDA MIX/VANC
Palm 32-128 32-128 32-128 64-128 64-128 64-128
Vancomycin 1-2 1-2 1-2 2-8 (16) 4-16 4-16 (32)
R E S I S TA N C E D E V E L O P M E N T
Biomaterials-associatedinfections
Potential of Immobilized Compounds to Induce Bacterial Resistance
No development of resistance
 Cells continuously exposed to surfaces functionalized with MIX
(palm+DNAse) exhibited the same susceptibility patterns to Palm (MIC
and MBC) as cells retrieved from control surfaces.
 Cells exposed to immobilized vancomycin exhibited the same MIC
value as cells in contact with control surfaces, but slight difference
were observed in the MBC values.
Heteroresistant or tolerant sub-
populations ?

22. Context/Motivation
Target
gene Direction Sequence(5’ à 3’)
Melting
Temperature
(°C)
Amplicon
size(bp)
Priming
efficiency
(%)
16S
F GGTCTTGCTGTCACTTATAGATGG 59.2
59.8
164 90.4
R CGGAAGATTCCCTACTGCTG
walkR
F TTGTCCGAAGATGAAGCAAG 59.0
59.9 248 93.7R CGCAGTAACGAACGACGATA
varsR
F CCGGCAATATAACCTGCACT 60.0
60.0
220 111.7
R GTAGTTGCGACGGATGAGGT
mdeA
F GCGAGAGGTGAAACGTTAGC 60.0
60.0
256 94.1
R AGAACAGAGCAGCAGCAACA
norA
F CCACCTGCTCCTACTACAAACA 59.0
60.0 212 99.1R ATGGAAAAGCCGTCAAGAGA
hla
F ACCGCCAATTTTTCCAGAAT 60.7
60.8
167 98.6
R CCTGGCCTTCAGCATTTAAG
clpP
F AACAACAAATCGCGGTGAAC 60.9
60.1
265 91.2
R TGCAGCCATACCGATACAAA
Reference gene
Transcriptomic analysis
R E S I S TA N C E D E V E L O P M E N T
Biomaterials-associatedinfections
Potential of Immobilized Compounds to Induce Bacterial Resistance
Vancomycin resistance
associated genes
Genes encoding Efflux
pumps
Virulence related Genes

23. Context/Motivation
walkR
varsR
norA
mdeA
hla
clpP
Log2 foldchange
UpregulatedDownregulated
walkR
varsR
norA
mdeA
hla
clpP
Transcriptomic analysis
R E S I S TA N C E D E V E L O P M E N T
Biomaterials-associatedinfections
Potential of Immobilized Compounds to Induce Bacterial Resistance
 Cells in contact with modified surfaces exhibited some genes involved in
microbial resistance and virulence equally or less expressed, as compared to the
ones recovered from control surfaces
No development of resistance

24. Context/Motivation
Macrophages-mediated phagocytosis
PDMS pDA MIX
S. aureus adhesion 4 h
Macrophages adhesion 2 h
100 µm
M A C R O P H A G E S A C T I O N
Biomaterials-associatedinfections
 Macrophages adhesion to unmodified PDMS tend to cluster, which may compromise their mobility and subsequently their
phagocytic activity.
 After pDA coating, macrophages were found more evenly distributed along these surfaces and a higher number of macrophages
seemed to adhere to the functional surfaces.
 Results suggest macrophages-mediated phagocytosis will be more efficient on the AMP functionalized PDMS.

25. Context/Motivation
Great potential to fight biomaterial-associated infections
Macrophages
clearance
No Resistance
More Susceptibility to
antibiotic treatment
Polydimethylsiloxane (PDMS)
C O N C L U S I O N S
Biomaterials-associatedinfections
 Co-immobilization of the AMP Palm and Dnase I, using pDA as an intermediate, holds great potential to fight
BAI, as remaining cells adhered to these surfaces did not develop resistance towards the immobilized
compounds, being even more susceptible to antibiotic therapy and the action of host immune system.

26. Context/MotivationA M P I M M O B I L I Z AT I O N
Biomaterials-associatedinfections
CK – Contact killing; IZ – inhibition zone
“+” visible growth
“-” lack of growth.
Presence (P) or absence (A) of inhibition zone
NT - not tested
PB – Polymixin B
PE – Polymixin E (Colistin)
E. coli P. mirabilis
CK IZ CK IZ
Strategy
[Antimicrobial]
(mg/mL)
0.5 1 0.5 1 0.5 1 0.5 1
2-step Palm - - A P + + NT NT
2-step MirG + + NT NT + - NT A
2-step MirK NT + NT NT NT + NT NT
2-step LAUR2 NT + NT NT NT + NT NT
2-step Temporin A NT + NT NT NT + NT NT
2-step PB - - A A + + NT NT
1-step PB - - A A + + NT NT
2-step PE - - ? ? NT NT NT NT
1-step PE - - ? ? NT NT NT NT
Controls CK IZ CK IZ
PDMS + NT + A
pDA + A + A
Contact-killing activity and leaching activity of unmodified PDMS surfaces, pDA-coated PDMS (pDA), pDA-coated PDMS surfaces
functionalized with different AMP via two-step approach (2-step) or one-step approach (1-step).
Urinary catheters functionalization
 Palm, Polimix B and E have effect in E.coli but not in P. mirablis
 MirG has effect on P. mirablis but not on E. coli
 No AMP is effective simultaneously in both bacteria
 Some AMP have contact-killing activity but some release occurs

27. Context/MotivationO V E R A L L C O N C L U S I O N S
 Good synergy outcomes in biofilm-associated infections
 Effective on resistant bacterial strains
 An added-value in the development of antimicrobial coatings
 No toxicity when immobilized
 No sign of resistance development
Can AMP contributing to fight resistance?
 Difficulty in achieving a broad-spectrum AMP
 AMP efficacy can be bacteria and strain dependent
• Difficulties in the control of polymicrobial biofilm- and biomaterials-associated infections
 Toxicity effects
 Lack of studies evaluating long term resistance development
 Costs
YES
But, it must be aware ……
Although promising, there is still a long way to go and important research to do!