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Peptides 49 (2013) 53– 58

Contents lists available at ScienceDirect

Peptides

jo ur nal homep age: www.elsev ier .com/ locate /pept ides

n vitro pharmacokinetics of antimicrobial cationic peptides alone andn combination with antibiotics against methicillin resistanttaphylococcus aureus biofilms

ibel Dosler ∗, Emel Mataraciepartment of Pharmaceutical Microbiology Faculty of Pharmacy, Istanbul University, 34116, Beyazit, Istanbul, Turkiye

r t i c l e i n f o

rticle history:eceived 3 June 2013eceived in revised form 7 August 2013ccepted 7 August 2013vailable online xxx

eywords:RSA

iofilmime kill curvesntimicrobial cationic peptidesntibiotic

a b s t r a c t

Antibiotic therapy for methicillin-resistant Staphylococcus aureus (MRSA) infections is becoming moredifficult in hospitals and communities because of strong biofilm-forming properties and multidrug resis-tance. Biofilm-associated MRSA is not affected by therapeutically achievable concentrations of antibiotics.Therefore, we investigated the in vitro pharmacokinetic activities of antimicrobial cationic peptides(AMPs; indolicidin, cecropin [1–7]-melittin A [2–9] amide [CAMA], and nisin), either alone or in com-bination with antibiotics (daptomycin, linezolid, teicoplanin, ciprofloxacin, and azithromycin), againststandard and 2 clinically obtained MRSA biofilms. The minimum inhibitory concentrations (MIC) and min-imum biofilm-eradication concentrations (MBEC) were determined by microbroth dilution technique.The time-kill curve (TKC) method was used to determine the bactericidal activities of the AMPs aloneand in combination with the antibiotics against standard and clinically obtained MRSA biofilms. The MICvalues of the AMPs and antibiotics ranged between 2 to 16 and 0.25 to 512 mg/L, and their MBEC values

were 640 and 512 to 5120 mg/L, respectively. The TKC studies demonstrated that synergistic interac-tions occurred most frequently when using nisin + daptomycin/ciprofloxacin, indolicidin + teicoplanin,and CAMA + ciprofloxacin combinations. No antagonism was observed with any combination. AMPsappear to be good candidates for the treatment of MRSA biofilms, as they act as both enhancers of anti-biofilm activities and help to prevent or delay the emergence of resistance when used either alone or incombination with antibiotics.

. Introduction

Staphylococcus aureus is a major human pathogen associatedith a variety of moderate to severe infections that present in the

ommunity as well as nosocomial settings. Methicillin-resistant S.ureus (MRSA) strains, which are now resistant to most antibiotics,re most often found in medical institutions, but are becomingncreasingly more associated with community-acquired infections21]. The danger of MRSA infections results from not only themergence of multidrug resistance, but also the ability of this bac-erium to produce biofilms. Kwon et al. [15] demonstrated thathe rate of biofilm positivity was significantly higher for MRSAhan methicillin susceptible strains. Staphylococci, especially MRSA

tains, are the most frequent cause of biofilm-associated infections,hich are a significant cause of morbidity and death and associ-

ted with indwelling medical devices [22]. Because the bacteria

∗ Corresponding author. Tel.: +90 212 440 02 57; fax: +90 212 440 02 57.E-mail addresses: sibeldosler@hotmail.com (S. Dosler),

melmataraci@hotmail.com (E. Mataraci).

196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.peptides.2013.08.008

© 2013 Elsevier Inc. All rights reserved.

in the biofilms become more resistant to antibiotic treatment andthe actions of the host immune system, the treatment of biofilm-associated infections is extremely difficult [17].

A biofilm is a microbial community that is attached to abi-otic surfaces and produced extracellular polysaccharides. It ischaracterized by the growth-dependent accumulation of multi-layered cell clusters that are in turn surrounded by an extracellularpolysaccharide matrix. The microbial cells that grow in biofilmsare physiologically distinct from the planktonic cells of the sameorganism. The bacteria in biofilms become more resistant toantibiotics and the host’s immune defense mechanisms as theyadapt to changing conditions together instead of as single cells[8,17].

Antimicrobial cationic peptides (AMPs) have attracted attentionas alternative antibiotics due to their prospective potency, rapidaction, and broad spectrum of activities against Gram-negativeand -positive bacteria, viruses, fungi and parasites. AMPs can be

found as a major component of the innate immune systems ofmost living organisms, including insects, plants, microorganisms,and mammals, to protect against environmental microorganisms.In addition, they exhibit multiple mechanisms of action and,

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onsequently, a low potential to induce de novo resistance, whichllows the limited use of other antibiotics [30].

Indolicidin is one of the shortest AMPs, a 13-residue tride-apeptide amide that is isolated from cytoplasmic granules ofovine neutrophils. It has an extremely high tryptophan con-ent and exhibits broad-spectrum antimicrobial and hemolyticctivities [25]. However, indolicidin’s activities differ from theell-defined channel-formation mechanism: instead, it createsores in cell membranes and induces the total disintegration ofembrane structures [12]. Cecropin (1–7)-melittin A (2–9) amide

CAMA) is a hybrid peptide that contains portions of the amino acidequences for silk moth peptide cecropin-A and bee venom pep-ide melittin. This hybrid forms ion-permeable channels in modelipid membranes and demonstrates improved antimicrobial activ-ties against Gram-positive bacteria, with a significant reductionn the toxicity that is typically observed with the use of melittin2,27]. Nisin is a 34-residue peptide that is isolated from the non-athogenic bacteria Lactococcus lactis, is one of the most extensivelytudied lantibiotics, possesses unusual amino acids (lanthioninend methyllanthionine residues), and belongs to a special groupf AMPs called bacteriocins. Nisin demonstrates rapid bacteri-idal activities against Gram-positive bacteria, including multidrugesistant pathogens, and is characterized by a dual mode of actiongainst cell membranes [24,31].

However, there are a lot of studies about in vitro activitiesf antimicrobial agents against biofilms [7,28]; the studies aboutharmacokinetic or pharmacodynamic picture of these activitiesre limited. We investigated the in vitro pharmacokinetic activi-ies of 3 AMPs – indolicidin, CAMA, and nisin – either alone or inombination with antibiotics (daptomycin, linezolid, teicoplanin,iprofloxacin, and azithromycin) which are routinely used in clinicsgainst standard and 2 clinically obtained MRSA biofilms.

. Materials and methods

.1. Bacterial strains

Two clinical isolates of MRSA were obtained from specimenshat were submitted to the Clinical Microbiology Laboratories ofstanbul University, Istanbul Faculty of Medicine. S. aureus ATCC3300 (Rockville, MD, USA) was the standard MRSA strain used inhis study. S. aureus ATCC 29213 (Rockville, MD, USA) was the qual-ty control strain used to verify the accuracy of the microdilutionest procedures for antibiotics.

.2. Antimicrobial substances

Two AMPs – CAMA and indolicidin – were obtained fromachem AG, and nisin was obtained from Sigma-Aldrich. Regardinghe antibiotics, daptomycin was provided by Novartis Pharma-euticals, teicoplanin, linezolid, and azithromycin were providedy Kocak Pharmaceutical Inc., and ciprofloxacin was provided byily Turkiye. Stock solutions from dry powders were prepared at

concentration of 1280 mg/L for the AMPs and 5120 mg/L for thentibiotics, and then stored at −80 ◦C for up to 6 months before use.

.3. Media

Tryptic soy broth supplemented with 1% glucose (TSB-glucose;ifco Laboratories) was used for biofilm production, cation-

djusted Mueller-Hinton broth (CAMHB, Difco Laboratories) wassed to determine the minimum inhibitory concentration (MIC),nd minimum biofilm-eradication concentration (MBEC) values,nd tryptic soy agar (TSA; Difco Laboratories) was used to

tides 49 (2013) 53– 58

determine the MBEC values and colony counts that were used inthe time-kill curve (TKC) analysis.

2.4. Determination of MIC

The MIC values of the antibiotics and AMPs were determinedusing microbroth dilution technique, as described by the Clinicaland Laboratory Standards Institute (CLSI) [5]. MIC was defined asthe lowest concentration of antibiotic that produced the completeinhibition of visible growth.

2.5. Biofilm formation

MRSA strains were cultured in 5 mL TSB-glucose for 24 h at 37 ◦Cwith 360◦ rotation (50 rpm) and diluted 1/50 in fresh TSB-glucose,yielding a final concentration of approximately 1 × 107 cfu/200 �L.This suspension was added to each well of a 96-well tissue cul-ture microtiter plate (Greiner), which was then incubated for 24 hat 37 ◦C. TSB-glucose was used as the negative control. After incu-bation, the waste media was gently aspirated, and the wells werewashed 3× with 250 �L phosphate buffered saline (PBS) solutionto remove any unattached bacteria and air-dried. Then, 200 �L 99%methanol was added to each well. The solution was allowed to fix-ate for 15 min, then it was aspirated and the plates were allowed todry. Wells were stained with 200 �L 0.1% crystal violet (in water)for 5 min. Excess stain was gently rinsed off with tap water, andthe plates were air-dried. The stain was re-solubilized by adding200 �L 95% ethanol and incubating the plate on an orbital shakerfor 30 min. The optical density was measured at 595 nm [10].

2.6. Determination of MBEC

Measurements of the antimicrobial susceptibilities of the MRSAbiofilms were assessed using an MBEC assay, which was performedas previously described with the following modifications [3]. The24 h biofilms in a 96 well tissue culture microtitre plates werewashed 3× with 250 �L PBS solutions and air-dried. Serial 2-folddilutions ranging from 640 to 0.06 mg/L for the AMPs and 5120 to5 mg/L for the antibiotics were prepared in CAMHB. Next, 200 �Lof each concentration was added to each corresponding well andplates were incubated 24 h at 37 ◦C. After incubation, the antibioticswere gently aspirated, the plates were washed 2× with sterile PBS,and the wells were thoroughly scraped with particular attentionto the edges of the wells. The contents of each well were removed,placed in 1 mL PBS, incubated in a sonicating water-bath (Bandelinsonopuls HD 2200) for 5 min to disrupt the biofilms, and 100 �Lsamples were plated on TSA. The colonies were counted after 24 h ofincubation at 37 ◦C. MBEC was defined as the lowest concentrationof AMP or antibiotic which bacteria fail to regrow after exposure tothe antimicrobial agents.

2.7. TKC determination

In order to assess the dynamic picture of the bactericidal activ-ities of AMPs, both alone and in combination with antibiotics, themodified TKC method was used [20]. 24-h biofilms were preparedin a 96-well tissue culture microtiter plate, washed 3× with 250 �LPBS, and air-dried. AMPs and antibiotics were diluted in CAMHBand added to each corresponding well, yielding a final concentra-tion of 1× MBEC, and the plates were incubated for 0, 2, 4, 7, or24 h at 37 ◦C. After incubation, the plates were washed 2× withsterile PBS, scraped and the contents were incubated in a sonicat-

ing water-bath, as described above. Following the disruption, serial1/10-fold dilutions were made and 100 �L samples were platedon TSA. Colonies were counted 24 h after incubation at 37 ◦C. Anantibiotic-free control of each strain was also included.

i / Peptides 49 (2013) 53– 58 55

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Table 2In vitro bactericidal activities of AMPs and antibiotics against biofilms of standardand clinical MRSA strains.

Antimicrobial agents Strains 3 log10 cfu/mL decrease from initialinoculum

2 h 4 h 7 h 24 h

Indolicidin Std − − − −CI 1 − − − −CI 2 − − − −

CAMA Std − + + −CI 1 − + − −CI 2 − + + −

Nisin Std − + + −CI 1 − + + +CI 2 − + + +

Daptomycin Std − + + −CI 1 − − − +CI 2 − − − +

Linezolid Std − − − −CI 1 − − − −CI 2 − − − −

Teicoplanin Std − − − −CI 1 − − − −CI 2 − − − −

Ciprofloxacin Std − − + −CI 1 − − − +CI 2 − − − −

Azithromycin Std − + + +CI 1 − − − +CI 2 − + + +

Std: MRSA ATCC 43300; CI 1: clinical isolate 1; CI 2: clinical isolate 2. The initialinoculum was ∼106 cfu/mL.

Table 3In vitro activities of AMP and antibiotic combinations against biofilms of standardand clinical MRSA strains.

AMP + antibiotic combinations MRSA Strains

Std CI 1 CI 2

Indolicidin+Daptomycin Syn Add Add+Linezolid Add Add Add+Teicoplanin Syn Syn Syn+Ciprofloxacin Syn Add Add+Azithromycin Add Add Add

S. Dosler, E. Matarac

TKCs were constructed by plotting mean colony countslog10 cfu/mL) versus time. The lower limit of detection for theime-kill assays was 2 log10 cfu/mL. The results were interpreted byomparing the effects of the various combinations and the effects ofhe most active agent alone. Synergy and antagonism were defineds a 2-log10 decrease or increase, respectively, in the colony countfter 24 h. Bactericidal activity was defined as a ≥3-log10 cfu/mLecrease from the initial inoculum.

.8. Statistically analysis

All experiments were performed in two independent assays. InIC, MBEC, and FICI determinations, when the results were dif-

erent in both experiments, we made another test for final result.ne way ANOVA-Bonferroni’s multiple comparison test was used

o compare differences between control and antimicrobials treatediofilms. P value < 0.001 was considering as statistically significant.

. Results

.1. Susceptibility

The in vitro activities of the studied AMPs and antibiotics againsttandard and clinically obtained MRSA planktonic cells or biofilmsre summarized in Table 1. According to this results, 3 AMPs –ndolicidin, CAMA and nisin – MIC values against MRSA planktonicells or those found in biofilms were 8–16, 4, and 2–8 mg/L, respec-ively, and all of their MBEC values were 640 mg/L. The MIC valuesf antibiotics – daptomycin, linezolid, teicoplanin, ciprofloxacin,nd azithromycin – were between 0.25 and 0.5, 1, 0.5 and 8, 1nd 16, and 256 and 512 mg/L, respectively. On the other hand,hen we assessed the anti-biofilm activities of these antibiotics,

he MBEC values were 1280–2560, 512–1280, 2560, 1280–2560,nd 1280–5120 mg/L, respectively. Throughout the study, the MICalues of the antibiotics against the quality control strain S. aureusTCC 29213 were within the accuracy range accepted by CLSI [6].

.2. Time-kill kinetics

The results of the TKC analysis show that 3-log10 killing againstRSA biofilms was determined within 4, 7 or 24 h using CAMA,

isin, daptomycin, and azithromycin alone, in addition to mostf the AMP and antibiotic combinations except those with line-olid (Table 2). As shown in Table 3, the TKC analysis also indicates

he enhanced killing of the tested strains when AMPs were usedn combination with all of the examined antibiotics, except line-olid. These synergistic interactions were seen in all of the MRSAiofilms, but most especially in nisin + daptomycin/ciprofloxacin,

able 1n vitro antibacterial and anti-biofilm activities of antimicrobial cationic peptidesnd antibiotics against standard and clinically obtained strains of MRSA.

Antimicrobial agent MIC (mg/L) MBEC (mg/L)

Std CI 1 CI 2 Std CI 1 CI 2

Cationic peptidesIndolicidin 16 8 8 640 640 640CAMA 4 4 4 640 640 640Nisin 8 2 2 640 640 640

AntibioticsDaptomycin 0.25 0.5 0.5 1280 1280 2560Linezolid 1 1 1 512 1280 640Teicoplanin 0.5 2 8 2560 2560 2560Ciprofloxacin 1 8 16 1280 2560 2560Azithromycin 512 256 512 5120 1280 2560

td: MRSA ATCC 43300 standard strain; CI 1: clinical isolate 1; CI 2: clinical isolate.

CAMA+Daptomycin Syn Add Add+Linezolid Add Add Add+Teicoplanin Syn Add Syn+Ciprofloxacin Syn Syn Syn+Azithromycin Add Syn Syn

Nisin+Daptomycin Syn Syn Syn+Linezolid Add Add Add+Teicoplanin Syn Add Add+Ciprofloxacin Syn Syn Syn+Azithromycin Add Syn Syn

Std: MRSA ATCC 43300; CI 1: clinical isolate 1; CI 2: clinical isolate 2; Syn: synergisticinteraction; Add: additive interaction.

indolicidin + teicoplanin, and CAMA + ciprofloxacin combinations(Figs. 1–3). No antagonism was observed with any combination.

4. Discussion

In our previous study, the 3 AMPs – indolicidin, CAMA and nisin– demonstrated in vitro activities with similar MIC, high-inoculumMIC, and MBEC values against standard MRSA ATCC 43300 and

56 S. Dosler, E. Mataraci / Peptides 49 (2013) 53– 58

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Fig. 1. Synergistic IND combinations that observed by time-kill determinationsagainst biofilms of (A) Standard MRSA ATCC 43300, (B) CI 1, or (C) CI 2 strains at 1×MBEC either alone or in combination with antibiotics. The X-axis represents time,and the Y-axis represents logarithmic MRSA survival. cfu: colony-forming unit; IND:ii

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Fig. 2. Synergistic CAMA combinations that observed by time-kill determinationsagainst biofilms of (A) Standard MRSA ATCC 43300, (B) CI 1, or (C) CI 2 strains at1× MBEC either alone or in combination with antibiotics. The X-axis representstime, and the Y-axis represents logarithmic MRSA survival. cfu: colony-forming unit;CAMA: cecropin (1–7)-melittin A (2–9) amide; DAP: daptomycin; TEC: teicoplanin;

ndolicidin; DAP: daptomycin; TEC: teicoplanin; CIP: ciprofloxacin; CI 1: clinicalsolate 1; CI 2: clinical isolate 2.

ot very high MBEC/MIC ratios which was obtained for antibi-tics (up to 8000-fold) [18]. In this study, we investigated then vitro activities of AMPs alone and in combination with antibioticsgainst standard and clinical MRSA planktonic cells or those foundn biofilms. However the planktonic MRSA cells were susceptible toll of the examined antibiotics, except azithromycin for standard,r ciprofloxacin and azithromycin for clinically obtained MRSA,he MBEC/MIC ratios of AMPs and antibiotics were 40–320 foldsnd 10–10.240 folds, respectively. Similar results were reportedn our previous study and have been noted by other researchers16,18,23,26].

Because of the mature biofilms are highly resistant to treat-ent with antimicrobial agents, the MBEC values of antibiotics are

xtremely higher than the MIC values. Clinically used antibioticsnd their dose regimens have classically developed to treat infec-ions due to the planktonic bacteria, so they are ineffective in theradication of biofilm based infections at the same doses. Thereforehe MBEC/MIC ratio is one of the important parameter for choos-ng the antibiotic in the treatment of biofilm associated infections.lthough the MIC values of the AMPs are not as low as the antibi-tics, it is notable that the MBEC/MIC ratios of the AMPs are fairly

ow in contrast to the examined antibiotics. Differences betweenhe activities of the antibiotics and AMPs might be due to theirissimilar structures and the mechanisms of the antibacterial

CIP: ciprofloxacin; AZM: azithyromycin; CI 1: clinical isolate 1; CI 2: clinical isolate2.

actions. The antimicrobial activities of several agents on a biofilmdepend on their molecular sizes, positive charges, permeabilitycoefficients, and bactericidal activities [14].

As seen in our results and reported in similar studies publishedby other groups, biofilm-associated bacteria are not affected bytherapeutically achievable concentrations of antimicrobial agentsfor many reasons, including decreased diffusion of antimicrobialagents into the extracellular matrix, increased activity of multidrugefflux pumps, quorum-sensing systems, antimicrobial tolerance,and the presence of persister and slow-growing cells [1,14]. Toovercome biofilm-associated resistance, the use of various com-binations of antimicrobial agents can provide synergistic effects,which in turn may rapidly enhance anti-biofilm activities and pre-vent or delay the emergence of resistance.

Cationic peptides have the ability to serve as anti-resistancecompounds, in addition to providing their own antimicrobialactivities [13]. Therefore, we attempted to determine if AMPsdemonstrate synergistic effects against MRSA biofilms and inwhat combinations. In our previous study [18], the microbroth

checkerboard technique demonstrated that synergistic interac-tions frequently result from almost all combinations of AMPsand antibiotics (except azithromycin) against MRSA ATCC 43300

S. Dosler, E. Mataraci / Pep

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Fig. 3. Synergistic NSN combinations that observed by time-kill determinationsagainst biofilms of (A) standard MRSA ATCC 43300, (B) CI 1, or (C) CI 2 strains at 1×MBEC either alone or in combination with antibiotics. The X-axis represents time,and the Y-axis represents logarithmic MRSA survival. cfu: colony-forming unit; NSN:nC

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isin; DAP: daptomycin; TEC: teicoplanin; CIP: ciprofloxacin; AZM: azithyromycin;I 1: clinical isolate 1; CI 2: clinical isolate 2.

iofilms, whereas most combinations generally demonstrate addi-ive effects against planktonic cells.

Although the determination of MIC is still the gold standard foretermining the activities of antimicrobial agents, and the micro-roth checkerboard technique is the most simple and widely usedechnique available for the assessment of antimicrobial combina-ions, these techniques do not provide any information about theime course of the antibiotic activities. This limitation can be over-ome with the use of TKC studies. In this study, the results of the TKCtudies demonstrate that the synergistic interactions against MRSATCC 43300 biofilms are present with all 3 AMP and antibioticombinations, except those with azithromycin. The only differenceetween these results is that the CAMA and nisin + linezolid com-inations were additive according to the TKC analyses, while theseombinations were synergistic according to the checkerboard tech-ique.

In combination studies, to determining the minimum bacteri-idal concentrations (MBC) of each drug alone or in combination,sing the “MBC checkerboard”, which is the extended method to

nvestigate bactericidal drug interactions by sampling from tubesr wells onto antibiotic-free media, could be possible. Therefore,

o predicting the synergism, high correlation between the TKC and

BC checkerboard results could be obtained rather than the clas-ic microbroth checkerboard results [11]. The difference about our

tides 49 (2013) 53– 58 57

combination results between checkerboard and TKC techniques,might be due to the using of classic microbroth checkerboard tech-nique.

The TKC results for the 2 clinically obtained MRSA biofilmsdemonstrates that synergism at 24 h can be obtained, especiallywhen nisin + daptomycin/ciprofloxacin, indolicidin + teicoplanin,and CAMA + ciprofloxacin combinations are used. On the otherhand, all 3 AMPs demonstrated only additive interactions whenused in combination with linezolid. No antagonism was observedwith any combination.

Daptomycin, teicoplanin and AMPs are members of differentclasses of antimicrobial agents that target the essential cell wallprecursors like lipid II. The molecular mechanism of action of theseantimicrobials is very different but starts from the same points.This molecular basis of mechanisms may explain how AMPs act insynergy with daptomycin or teicoplanin. Linezolid, azithromycinand ciprofloxacin show their activities against bacteria via inter-acting with the intracellular mechanisms like inhibition of proteinsynthesis or DNA gyrase enzyme. By this way disturbing the bac-terial cellular membranes, AMPs allow the increase of intracellularuptake of these antibiotics more easily. The synergistic interactionswith AMPs and ciprofloxacin might be due to the increased accessto the cytoplasmic membrane through cationic peptides, follow-ing breakdown of peptidoglycan. On the other hand, as seen withthe linezolid or azithromycin and AMP combinations, not only apermeability-increasing effect of AMPs is likely to be the main rea-son for synergistic or additive interactions but also it may be theresult of a combined effect of access to the intracellular targets forantibiotics and AMPs itself [19,29].

The results of the TKC analysis also demonstrate that CAMA,nisin, daptomycin, azithromycin, and most of the AMP and antibi-otic combinations are rapidly bactericidal against all of the studiedMRSA strains within 4, 7 or 24 h. Our results demonstrate that 3-log10 killing of the standard and clinically obtained MRSA biofilmsoccurred even within 4 h following the administration of CAMA,nisin and most of their combinations. A previous study indicatedthat nisin alone or in combination with antibiotics demonstratesvery effective and rapid antimicrobial activities against clinicallyobtained methicillin susceptible S. aureus and MRSA planktoniccells within 4 h, independent of whether or not the bacteria areresistant to antibiotics [9].

In conclusion, biofilm-associated bacteria are not affected bytherapeutically achievable concentrations of antimicrobial agents,because of the mechanisms involved in eradication or inhibition ofbiofilms are very different. The assessment of the anti-biofilm activ-ities of antimicrobial agents is generally based on interference withquorum sensing, inhibition of adhesion, enhancement of disper-sion, and various experimental and promising alternatives, such asthe use of biofilm-specific antibodies, bacteriophage-based treat-ments, and the species-specific control of biofilms [4]. Even thoughwe know that larger studies are needed to assess with greater cer-tainty, according to the results of this study, AMPs appear to begood candidates for the treatment of MRSA biofilms because theyboth enhance anti-biofilm activities and preventing or delaying theemergence of resistance when used either alone or in combinationwith antibiotics.

Acknowledgement

This work was supported by the Research Fund of Istanbul Uni-versity (Project No.: 21655).

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