Antibacterial Activities of Lipopeptide (C10)

19 Pages·2017·2.16 MB·English

International Journal o f
Molecular Sciences
Article
Antibacterial Activities of Lipopeptide
(C ) -KKKK-NH Applied Alone and in
10 2 2
Combination with Lens Liquids to Fight Biofilms
Formed on Polystyrene Surfaces and Contact Lenses
MalgorzataAnnaPaduszynska1,*,MagdalenaMaciejewska1,2,KatarzynaEwaGreber3,
WieslawSawicki3andWojciechKamysz1
1 DepartmentofInorganicChemistry,FacultyofPharmacy,MedicalUniversityofGdansk,
80-416Gdansk,Poland;[email protected](M.M.);[email protected](W.K.)
2 PharmaceuticalLaboratoryAvenaSp.z.o.o.,86-031Osielsko,Poland
3 DepartmentofPhysicalChemistry,FacultyofPharmacy,MedicalUniversityofGdansk,
80-416Gdansk,Poland;[email protected](K.E.G.);[email protected](W.S.)
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:11December2018;Accepted:14January2019;Published:17January2019
Abstract: The widespread use of biomaterials such as contact lenses is associated with the
developmentofbiofilm-relatedinfectionswhichareverydifficulttomanagewithstandardtherapies.
Theformationofbacterialbiofilmsonthesurfaceofbiomaterialsisassociatedwithincreasedantibiotic
resistance. Owing to their promising antimicrobial potential, lipopeptides are being intensively
investigated as novel antimicrobials. However, due to the relatively high toxicity exhibited by
numerouscompounds,alotofattentionisbeingpaidtodesigningnewlipopeptideswithoptimal
biological activities. The principal aim of this study was to evaluate the potential ophthalmic
applicationoflipopeptide(C ) -KKKK-NH . ThislipopeptidewassynthesizedaccordingtoFmoc
10 2 2
chemistryusingthesolid-phasemethod. Theantibiofilmactivitiesofthelipopeptide,antibiotics
usedinocularinfections,andcommerciallyavailablelensliquidsweredeterminedusingthebroth
dilution method on polystyrene 96-well plates and contact lenses. Resazurin was applied as the
cell-viabilityreagent. Theeffectivenessofthecommerciallyavailablelensliquidssupplementedwith
thelipopeptidewasevaluatedusingthesamemethodandmaterials. (C ) -KKKK-NH exhibited
10 2 2
strongeranti-biofilmpropertiescomparedtothoseofthetestedconventionalantimicrobialsand
showedtheabilitytoenhancetheactivityoflensliquidsatrelativelylowconcentrations(4–32mg/L).
EstimationoftheeyeirritationpotentialofthelipopeptideusingToxtreesoftware2.6.13suggests
thatthecompoundcouldbesafelyappliedonthehumaneye. Theresultsofperformedexperiments
encouragefurtherstudieson(C ) -KKKK-NH anditspotentialapplicationintheprophylaxisof
10 2 2
contactlens-relatedeyeinfections.
Keywords: lipopeptides;biofilm;persistercells;ocularinfections;biofilmoncontactlenses
1. Introduction
Nowadays, the alarming growth and spread of antibiotic-resistant microorganisms is such a
seriousproblemthatitthreatenstheachievementsofmodernmedicine[1]. Moreover,microorganisms
formbiofilmsonthesurfaceofbiomaterialsorhumantissuesthatareup1000timesmoreresistant
tostandardantibiotictherapycomparedtotheirplanktoniccounterparts[2,3]. Thecontinuingrise
inantibioticandmulti-drugresistantaswellasbiofilm-relatedbacterialinfectionsisamajorglobal
medicalhealthissueandisassociatedwiththefailureofclinicaltreatment,thelimitationofantibiotic
Int.J.Mol.Sci.2019,20,393;doi:10.3390/ijms20020393 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2019,20,393 2of19
use,andincreasedmorbidity,mortality,andhealthcarecosts. Allthesefactorssignificantlyimpactthe
worldeconomyand,therefore,anewgenerationofantimicrobialcompoundsisrequired[4,5].
Cationicantimicrobialpeptides(AMPs)arepromisingalternativestoconventionalantibiotics,
duetotheiruniquemechanismofactionthatreducestheriskofbacteriadevelopingresistanceto
them,andalsobecauseoftheirabilitytoinhibitmulti-drugresistantbacterialbiofilms[6,7]. AMPs,
aspartoftheinnateimmunesystem,naturallyoccurinmanypartsofthehumanbody. Forinstance,
LL-37, defensins and psoriasin, as part of tear fluid, form an important part of the innate defense
systeminthehumaneye[8]. LL-37isanintensivelystudiedhumanAMPwithconfirmedanti-biofilm
activity[9,10]thathasprovidedafoundationonwhichtodesignnumerouspeptidestofightbacterial
biofilm [11,12]. Natural AMPs, as wellas their derivatives, have been investigatedwith regard to
theirpotentialophthalmicuse[13,14],includingtopicalapplication[15,16],incorporationintocontact
lenses(CLs)[17,18],andaspreservativeagentsinCLsolution[14,19]andcornealstoragemedia[20].
However,despitethepromisingresultsobtainedwithAMPs,theirbroadspectrumofantimicrobial
activityandtheirlowriskofresistancedevelopment,theapplicationofthesecompoundsintherapyis
limitedduetotheirpotentialtoxicity,allergenicity,enzymaticdegradation,poorstabilityinvivo,and
highcostsofproduction[21–26].
TheresearchonfeaturesdeterminingtheantimicrobialactivityofAMPshaveyieldedessential
informationforthedesignofnovel,highlyeffectivecompoundswithoptimizedbiologicalproperties
that can also be produced at lower cost compared to their natural antimicrobial counterparts.
Numerousstudiesfocusonevaluatinganddesigningshorteranalogs,creatingmultimericAMP-based
sequencesanddevelopingpeptidomimeticswhichcanimitatethebactericidalmechanismofaction.
Asuccessfulapproachtomodulatingtheactivityandbioavailabilityofpeptidesistheacylation
of cationic peptides with fatty acid. It has been shown that the introduction of D-amino acid or
non-peptideresiduessignificantlyimprovestheantimicrobialspectrumactivityofcationicpeptides
anddeterminesahigherresistancetoproteolyticdegradation[27,28]. Simplemodification,suchas
the acylation of short cationic residue, has resulted in short synthetic lipopeptides, a particularly
promising group of compounds exhibiting a strong and broad spectrum of antimicrobial activity.
They are composed of short positively-charged peptide chains conjugated with a fatty acid that
providesamphipathicity. Thosetwofeaturesdeterminethesurface-activepropertiesofthecompounds
andallowthemtoelectrostaticallyinteractwithanegatively-chargedmicrobialmembrane,leadingto
arapid-killdrug-resistantpathogen[29]. Thecompoundsarecost-effectiveandlesstime-consuming
toproduceincomparisonwithnativeAMPs.
Sofar,researchhasallowednumerousshortlipopeptidesendowedwithhighantibacterialaswellas
antifungalactivitytobeidentified[21,30–32]. Theselipopeptideshavealsobeenfoundtobeeffective
againstbiofilmsandmulti-drugresistantbacteria[33]. However,theirpracticaluseinophthalmology
remainslimited. Twocriticalissuesaretheirpotentialtoxicityorallergenicity[31,34]. Theseissuesare
especiallyimportantinthecaseofsuchadelicateandsensitivestructurelikethehumaneyeand,therefore,
agreatdealofattentionisbeingpaidtooptimizingthebiologicalactivitiesoflipopeptides.
In previous studies, we identified very a promising compound—(C ) -KKKK-NH —which
10 2 2
exhibitsstrongantibacterialactivitiesandlowtoxicitytowardshumancellsinvitro[35,36]. Inthis
study we have further investigated the antimicrobial activity of this compound with regard to its
potentialapplicationinophthalmology,andpre-evaluateditsirritationpotentialviacomputational
methods which have proved to be very useful in predicting and describing the properties of the
compound[37–39].
2. Results
2.1. ActivityoftheLipopeptideandConventionalAntibioticsagainstBiofilmsFormedonPolystyrene
Thetestedcompoundsexhibiteddiverseantibiofilmactivitiestowardsvariousbacterialspecies.
The durability of the antimicrobial effect after the withdrawal of the active compound varied
Int.J.Mol.Sci.2019,20,393 3of19
significantlydependingontheappliedcompoundandthetestedstrain. Inmanycases,theremovalof
theantibioticcausedpartialorevenfullrenewalofbacterialbiofilms.
StructuresformedbyStaphylococcusepidermidis(SE)onthesurfaceof96-wellplatesturnedoutto
besensitivetoalltestedcompounds(Figure1,Table1). Theapplicationofsolutionsofciprofloxacin
significantlyreducedthemetabolicactivityofcellsinthepre-grownstructures. Theantibioticusedat
Int. J. Mol. Sci. 2019, 20, x 5 of 20
arangeofconcentrationsfrom1–8mg/Lcausedaca. 70–80%decreaseinthemetabolicactivityof
culturedbacteria,whileco ncentrationsof16mgP/sLeuadnodmhoingahse arerreusguinltoesda inthereductionofmetabolic
activityto10%andCloipwroefrloinxaccoinm pariso≤n1 tothepos3it2i vecontrol≤.1A dditiona1l6in cubationinthepure
medium after theCrhelmoroamvaplhoenfiscooll ution1s28o f ciprofl>o2x5a6c in (1–1284 mg/L) ca2u5s6e d an increase in the
metabolicactivityofthNeeboamcytecrinia (Table1)6.4O nlythec>o25n6c entration8o f256mg/L12o8f theantibioticcreated
Lipopeptide 256 >256 64 64
apermanentantibiofilmeffect.

Figure 1. Activity of the lipopeptide and conventional antimicrobials applied at concentrations
Figure 1. Activity of the lipopeptide and conventional antimicrobials applied at concentrations of 1-
of 1–256 mg/L against SE biofilms formed on polystyrene (A) results read after 24 h exposure to
256 mg/L against SE biofilms formed on polystyrene (A) results read after 24 h exposure to
compounds; and (B) results read after the withdrawal of compounds and an additional 24 h of
icnocmubpaotuionndisn; ManHd B(BII). Trehseurletss urletasda raefpterre stehnet ewdiathsdthraewpaelr coefn tcaogmepoofumnedtas baonlidc aacnt ivaidtydiitniocnoaml p2a4r ihs oonf
tioncpuobsaittiivoen( 1i0n0 %M)HanBd InIe. gTahtiev er(e0s%ul)tcso nartreo lps;rResSeDnt≤ed1 5a%s .the percentage of metabolic activity in
comparison to positive (100%) and negative (0%) controls; RSD ≤ 15%.
Int.J.Mol.Sci.2019,20,393 4of19
Table1. Activitiesofconventionalantimicrobialsandthelipopeptide—(C ) -KKKK-NH against
10 2 2
bacterialbiofilmsformedonpolystyreneplatespresentedasMBEC—minimumbiofilmeradication
concentration(mg/L);MBEC90—thelowestconcentrationwhichallowedtoreducethemetabolic
activity of bacteria by at least 90 ± 5%; MBEC 90 II – the lowest concentration which resulted in
permanentreductionofmetabolicactivitybyatlast90±5%;MBEC50—thelowestconcentrationwhich
allowedtoreducethemetabolicactivitybyatleast50±5%;MBEC50II—thelowestconcentration
resultedinpermanentreductionofthemetabolicactivitybyatleast50±5%.
Compound MBEC90 MBECII90 MBEC50 MBECII50
Staphylococcusepidermidis
Ciprofloxacin 16 256 ≤1 128
Chloramphenicol 256 >256 32 >256
Neomycin 16 16 ≤1 8
Lipopeptide 16 16 16 16
Staphylococcusaureus
Ciprofloxacin >256 >256 16 128
Chloramphenicol >256 >256 128 >256
Neomycin 64 64 4 64
Lipopeptide 32 32 8 16
Enterococcusfeacalis
Ciprofloxacin >256 >256 64 256
Chloramphenicol >256 >256 32 >256
Neomycin >256 >256 64 >256
Lipopeptide 32 32 16 32
Escherichiacoli
Ciprofloxacin 32 32 ≤1 ≤1
Chloramphenicol 16 >256 8 >256
Neomycin >256 >256 8 >256
Lipopeptide 64 64 64 64
Pseudomonasaeruginosa
Ciprofloxacin ≤1 32 ≤1 16
Chloramphenicol 128 >256 4 256
Neomycin 64 >256 8 128
Lipopeptide 256 >256 64 64
Similar antibiofilm activity against SE was presented by neomycin. However, in the case of
thisantibiotic,theeffectremainedafteritswithdrawalandadditionalincubation. Chloramphenicol
exhibitedratherlowantibiofilmpotential. Inthefirstantibiofilmassay,thecompoundreducedthe
metabolicactivityofbacteriatoca. 12,20,and25%onceappliedatconcentrationsof256,128,and
64mg/Lrespectively. Theactivitywasremovedtotallyinthesecondassay—themetabolicactivity
of bacteria in all the samples after exposure and the subsequent withdrawal of chloramphenicol
increasedsignificantly. Lipopeptide(C ) -KKKK-NH turnedouttobeveryactiveagainstSEbiofilm.
10 2 2
Applicationatconcentrationsof16–256mg/Lcausedthemetabolicactivityofbacterialcellstoreduce
toca. 5%. ThiswasthestrongestreductionofmetabolicactivityofSEcellsobservedinthisassay.
Moreover,theeffectremainedafteradditionalincubationinthepuremediumafterthewithdrawalof
thelipopeptide.
Staphylococcus aureus (SA) cultured on polystyrene plates turned out to be less sensitive in
comparison to SE (Figure 2, Table 1). The difference in susceptibility is especially visible in the
caseofconventionalantibiotics. CiprofloxacinreducedthemetabolicactivityofSAtolessthan20%
whenappliedatthehighesttestedconcentrationandtoca. 30%whenappliedatconcentrationsof
128–164mg/L.Theantibiofilmeffectwaspermanentonlyatthetwohighestconcentrations(Table1).
TheremovaloftheantibioticSAinthesampletreatedwithaconcentrationof64mg/Lincreased
metabolic activity to ca. 70% of the positive control. Neomycin exhibited some higher and more
permanentactivity. However,thereductionofmetabolicactivitywasnotassignificantasinthecaseof
SE.Themostpotentantistaphylococcalagentwasthelipopeptide. Thecompoundcausedareduction
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inthemetabolicactivityofSAcellsbyover90%whenappliedatconcentrationsof32mg/Landhigher.
Theexposuretothelipopeptidecausedapermanentantibiofilmeffect—metabolicactivitydidnot
increaseafterthecompoundwasreplacedwithpureMHBII.AsinthecaseofSE,chloramphenicolwas
theleastpromisingagent. ThemetabolicactivityofSAwasreducedbyhalfonlyaftertheapplication
ofthecompoundatconcentrationsof128–256mg/Landincreasedsignificantlyafterthewithdrawal
Int. J. Mol. Sci. 2019, 20, x 6 of 20
oftheantibiotic.

Figure 2. Activity of the lipopeptide and conventional antimicrobials applied at concentrations
Figure 2. Activity of the lipopeptide and conventional antimicrobials applied at concentrations of 1–
of1–256mg/LagainstSAbiofilmsformedonpolystyrene(A)resultsreadafter24hexposureto
c2o5m6 pmougn/Ld sa;gaanindst( BS)Ar ebsuioltfsilmresa dfoarmfteerdt hoen wpiothlydsrtaywreanleo (fAc)o mrepsouultns drseaadn dafatner a2d4d ihti oenxaplo2s4urhe otfo
icnocmubpaotuionndisn; ManHdB (BII). Trheseurletssu rletsadar eafpterer stehnet ewdiatshdthreawpearl coenf tcaogmeopfomunedtasb aonlidc aacnti vaidtydiitniocnoaml p2a4r ihso onf
tionpcuobsiattiivoen( 1i0n0 %M)HanBd nIIe. gTathive er(e0s%u)ltcso natrreo lsp;rResSeDnt≤ed1 5a%s .the percentage of metabolic activity in
comparison to positive (100%) and negative (0%) controls; RSD ≤ 15%.
BiofilmsformedbyEnterococcusfeacalis(EF)turnedouttobethemostresistanttoconventional
antimicrobials(Figure3,Table1). Applicationofallthreecompoundsatthehighestconcentrations
resultedinareductionofmetabolicactivityinbiofilmstoca. 35%ofinitialpopulations. Afterthe
withdrawalofantibiotics,theeffectremainedforciprofloxacin,whiletheremovalofchloramphenicol
andneomycinresultedinthecompleterenewalofthemetabolicactivityofbacteria. Thelipopeptide
exhibitedtheabilitytopermanentlyeradicatethebiofilmatconcentrationsof32–256mg/L.Inboth
antibiofilmassays,areductionofthemetabolicactivityofEFtolessthan10%ofthepositivecontrol
wasobserved.
IInntt.. JJ.. MMooll.. SSccii.. 22001199,, 2200,, x3 93 76 ooff 2109

Figure 3. Activity of the lipopeptide and conventional antimicrobials applied at concentrations
Figure 3. Activity of the lipopeptide and conventional antimicrobials applied at concentrations of 1–
of 1–256 mg/L against EF biofilms formed on polystyrene (A) results read after 24 h exposure to
256 mg/L against EF biofilms formed on polystyrene (A) results read after 24 h exposure to
compounds; and (B) results read after the withdrawal of compounds and an additional 24 h of
compounds; and (B) results read after the withdrawal of compounds and an additional 24 h of
incubationinMHBII.Theresultsarepresentedasthepercentageofmetabolicactivityincomparison
incubation in MHB II. The results are presented as the percentage of metabolic activity in
topositive(100%)andnegative(0%)controls;RSD≤15%.
comparison to positive (100%) and negative (0%) controls; RSD ≤ 15%.
Some higher concentrations of the lipopeptide were required to fight structures formed by
Escherichiacoli(EC)(Figure4,Table1). Thiseffectwasobservedafterapplicationofthelipopeptide
atconcentrationsof64mg/Landhigher. However,thereductionofmetabolismwasalsoveryhigh
anddidnotdeteriorateafterthewithdrawalofthecompound. Asimilareffectwasobservedafter
theexposureofECbiofilmstociprofloxacinatconcentrationsof32–256mg/L.Treatmentwithlower
concentrations reduced metabolic activity by 75 to 85%, however, after removal of the antibiotic,
acertainincreaseofmetabolicactivitywasobserved. Chloramphenicol,aswellasneomycin,also
exhibitedratherhigheffectivenesstowardsECbiofilms;however,thebacterialpopulationswereable
tofullyrestoretheirmetabolicactivitywhenthecompoundswereremovedfromtheenvironment.
Int.J.Mol.Sci.2019,20,393 7of19
Int. J. Mol. Sci. 2019, 20, x 8 of 20

Figure 4. Activity of the lipopeptide and conventional antimicrobials applied at concentrations
Figure 4. Activity of the lipopeptide and conventional antimicrobials applied at concentrations of 1–
of1–256mg/LagainstECbiofilmsformedonpolystyrene(A)resultsreadafter24hexposureto
c2o5m6 pmoug/nLd sa;gaanidns(tB )ErCes ublitosfrilemads affotermrtehde wonit hpdoralywsatylroefnceo m(Ap)o urnesduslatsn dreaaddd iatifotenra l2244 hh oefxipnocusubraeti otno
icnomMpHoBunIId.sT;h aenrdes u(Blt)s arreesuplrtess ernetaedd aasfttehr etpheer cwenitthadgeraowfaml eotaf bcoolimcpacotuivnidtys inancdo mapdadriitsioonnatlo p24o shit ivoef
(i1n0c0u%ba)taionnd nineg aMtiHveB( 0I%I. )cTohnet rorelss;uRltSsD a≤re 15p%re.sented as the percentage of metabolic activity in
comparison to positive (100%) and negative (0%) controls; RSD ≤ 15%.
Pseudomonasaeruginosa(PA)formedabiofilmwhichexhibitedthehighestresistancetowardsthe
lipopeptide(Figure5,Table1). Thecompoundreducedthemetabolicactivityofbacteriato10%only
whenappliedataconcentrationof256mg/L.Unfortunately,thebacteriarepopulatedandgained
40%ofthemetabolicactivityofthepositivecontrolaftertheremovalofthelipopeptide. Application
oflowerconcentrationsresultedina50%decreaseofbacterialmetabolismwhichdidnotincrease
afterthewithdrawalofthecompound. CiprofloxacinwashighlyactiveagainstPA—itreducedthe
metabolicactivityofbacteriabymorethan90%evenatthelowestappliedconcentrations. Further
incubationinthemediumwithoutantibioticscausedacertainrenewalofmetabolicactivitywithinthe
biofilm,butonlyinwellstreatedwiththeantibioticappliedatconcentrationslowerthan32mg/L.
ExposureofthePAbiofilmtochloramphenicolatconcentrationsof128–256and32–64mg/Lresulted
inthereductionofthemetabolicactivitiesofbacteriabyca. 90%and70%,respectively. Theincubation
ofPAafterreplacingsolutionsofchloramphenicolwithMHBIIresultedinasignificantincreasein
metabolism. Interestingly,pretreatingthebiofilmwiththecompoundatconcentrationslowerthan
32mg/Lresultedinasignificantpromotionofbiofilmgrowth. Verysimilarresultswereobtainedfor
neomycin. Thecompoundreducedmetabolicactivitybyover90%atconcentrationsof64–256mg/L
and75%aftertheexposureofPAtothecompoundataconcentrationof32mg/L.Removalofthe
antibiotic resulted in a significant increase of PA metabolism. As in the case of chloramphenicol,
pretreatmentofaPAbiofilmwithconcentrationslowerthan32mg/Lcausedtheenhancedmetabolism
ofbacteriaincomparisontothepositivecontrol.
IInntt.. JJ.. MMooll.. SSccii.. 22001199,, 2200,, x3 93 98 ooff 2109

Figure 5. Activity of the lipopeptide and conventional antimicrobials applied at concentrations
Figure 5. Activity of the lipopeptide and conventional antimicrobials applied at concentrations of 1–
of1–256mg/LagainstPAbiofilmsformedonpolystyrene(A)resultsreadafter24hexposureto
256 mg/L against PA biofilms formed on polystyrene (A) results read after 24 h exposure to
compounds;and(B)resultsreadafterthewithdrawalofcompoundsandadditional24hofincubation
compounds; and (B) results read after the withdrawal of compounds and additional 24 h of
inMHBII).Theresultsarepresentedasthepercentageofmetabolicactivityincomparisontopositive
incubation in MHB II). The results are presented as the percentage of metabolic activity in
(100%)andnegative(0%)controls;RSD≤15%.
comparison to positive (100%) and negative (0%) controls; RSD ≤ 15%.
2.2. ActivityofLipopeptideandCLSolutionsagainstBiofilmsFormedonCLs
2.2. Activity of Lipopeptide and CL Solutions against Biofilms Formed on CLs
Commercially-availablelensliquidsprovedtobehighlyactiveagainstbiofilmsformedonCLs.
TheyCroemdumceedrcitahlelym-aevtaaiblaoblilsem leonfs blaiqcuteirdisa pcuroltvuerded too nbeC Lhsigbhylya tacletiavset 9a0g%ainfostr bthioefivlmasst mfoarjmoreidty oonf
CteLstse. dTshterya irnesd.uBcoetdh ltihqeu midestAabaonlidsmB coaf ubsaecdtearirae dcuulcttuiorendo ofnb aCctLesr ibayl mate lteaabsotl i9s0m%t ofo1r0 t%heo vfathste mpoasjoitriivtye
ocof ntetrsoteld(o rstlroawinesr. )BfoorthS Aliq,SuEid,sE FA, aannddE BC ,cwauhsieled oan lryedliuqcutiidonA odf ebmacotnesrtiaral tmedettahbisolaicstmiv ittoy 1a0g%ai nosft tPhAe.
Tpohseitaipvpe lcicoanttiroonl o(ofrt hloewlipero)p feopr tiSdAe, dSiEss, oElFv,e danidn PEBCS, awllhoiwle eodnblyio lfiiqlmuisdf oAr mdeemdobnysatrllatteedst etdhisst raacitnivsittyo
abgearienmsto PvAed. fTrhoem atphpelCicLatsi.oTnh oefh tihgeh elsiptoepffeepcttiivdeen desissswolavsedo bisne rPvBeSd faollroSwEeadn bdioEfFi.lmAsf teforremxpedo sbuyre atlol
ttehsetelidp ostpreapintsid teo abtea rceomnocvenedtr afrtioomn othfe8 CmLgs/. LT,hteh ehimgheetastb oeflifcecatcivtievniteysso wfbaasc otebrsiearrveeddu fcoerd SbEy aantdl eEaFst.
A90f%te.r Feoxrpsousuchrea tsoi gtnhiefi cliapnotpdeepctrideaes eato af mcoentacbenotlirsamtioonf SoAf 8c emllgs,/Lth, ethaep pmliectaatbioonlico fatchtieviltiyp oopfe bpaticdteeraiat
raecdouncceedn btrya taito lneaosft 1960%m.g F/oLr wsuacsh nae seidgendif.icTahnet dmeocrsetadsief fiocfu mltectuabltoulrisemst oofe SliAm cinelalste, twheit ahplpipliocpateipotni doef
tshoelu ltiipoonpsewpteidree EatC a acnodncPeAnt—rattoiorne douf c1e6 tmheg/mL ewtaabs onliecedaecdti.v Tithyeo mf tohset sdeifsftircauilnt scublytumreos rteo tehlaimni9n0a%te,
wacitohn cliepnotpraetpiotindoe fs3o2lumtigo/nLs owfelripe oEpCep atnidde PwAe—retroe qruediruedce( Fthigeu mree6t)a.bolic activity of these strains by
moreT thheasne 9v0e%ry, ap croonmceisnintrgatrioesnu oltfs 3w2 merge/Lob otfa ilnipeodpwephtiedne twheereex rpeoqsuuirreedt o(FCigLurlieq 6u)i.d s and solutions
of lipTohpeespet videeryl apsrteodmuisnintigl trheseurletsa dwinerge oofbrteasiuneltds. wOhnecne tthhee eCxpLolsiquureid tso wCLer eliqruemidos vaendd asnodlutthioenCs Losf
wlipeorepefuprttidhee rlainstceudb autnetdil itnheM rHeaBdIiIn,gt hoef mreestualbtso. lOicnaccet itvhitei eCsLo flibqaucidtesr iwalepreo preumlaotivoends aonfdth tehem CaLjosr iwtyeoref
fsutrratihnesr winecreubnaetaerdly ifnu lMlyHreBn eIwI, etdh.eT mheetaanbtoibliacc taecrtiaivlietfifeesc toof fbCaLctseorilault iponopsurelamtiaoinnes doof ntlhyef omraSjEo,riwtyh iolef
sfotrraliinqsu wideAre annedartlhye fEuFllys trreanine,wbeiodfi. lTmheg raonwtitbhacintecrrieaal seefdfe.cSt oomf eCbLe tstoelru,tbiountsa lrseomnaointefudl loynslayt ifsofry iSnEg,,
while for liquid A and the EF strain, biofilm growth increased. Some better, but also not fully
satisfying, results were obtained for the lipopeptide. It permanently removed SA and SE biofilms
Int.J.Mol.Sci.2019,20,393 9of19
Int. J. Mol. Sci. 2019, 20, x 10 of 20
resultswereobtainedforthelipopeptide. ItpermanentlyremovedSAandSEbiofilmsfromCLsonce
from CLs once applied at a concentration of 16 mg/L. For Gram-negative strains, the bacterial
appliedataconcentrationof16mg/L.ForGram-negativestrains,thebacterialpopulationsregrewto
populations regrew to a high extent (PA was fully restored, EC to ca. 50%) after the removal of the
ahighextent(PAwasfullyrestored,ECtoca. 50%)aftertheremovalofthelipopeptideatalltested
lipopeptide at all tested concentrations. Similarly as in the case of CL solution A, treating EF with
concentrations. SimilarlyasinthecaseofCLsolutionA,treatingEFwiththelipopeptidepromoted
the lipopeptide promoted bacterial growth.
bacterialgrowth.

Figure6.Activityofthelipopeptideappliedatconcentrations4–128mg/Lagainstbiofilmsformedon
Figure 6. Activity of the lipopeptide applied at concentrations 4–128 mg/L against biofilms formed
CLs.Theresultsarepresentedasthepercentageofmetabolicactivityincomparisontopositive(100%)
on CLs. The results are presented as the percentage of metabolic activity in comparison to positive
andnegative(0%)controls;RSD≤20%.
(100%) and negative (0%) controls; RSD ≤ 20%.
2.3. AntibiofilmActivityoftheLipopeptideAppliedinCombinationwithCLLiquids
2.3. Antibiofilm Activity of the Lipopeptide Applied in Combination with CL Liquids
2.3.1. BiofilmsFormedonPolystyreneSurfaces
2.3.1. Biofilms Formed on Polystyrene Surfaces
In this assay we found that the supplementation of CL solutions with lipopeptide positively
influenIcne dthtihs eaisrsaanyt iwbaec tfeoruinalda tchtiavti ttyhe( Fsiugpuprele7m).eTnthaitsiowna osfe sCpLe csioallulytionnosti cweiatbhl elifpoorpleiqputiiddeB p,owsihtiivchely
deinmfolunesntrcaetde dthleoiwr aenrteibffaecctteirvieanl eascstivinityc o(mFigpuarries o7n). Ttohilsiq wuiads eAs.pTechiealllya tnteorticcaeuabseled ftohre lirqeudiudc Bti,o wnhoifch
mdeteamboolnicstarcattievdit yloowfbera ceteffreiactbivyemneosrse itnh acno9m0p%arfoisroanl mtoo sltiqaullitde sAte.d Tshtrea ilnast,tebru tcasuupsepdle mtheen rtaetdiounctwiointh of
thmeleitpaobpoelpict iadcetirveistuy ltoefd biancatenrieav ebny hmigohreer tdheacnr e9a0s%e offomr aiclmroobsiat lamll etteasbteodli ssmtr.aEinxsp, obsuutr esuopfpPAlembieonfitlamtison
towliiqtuhi dthAe lriepdoupceepdtibdieo firlemsumlteedta bino laisnm evtoenc ah.i1g5h%ero dfethcreepaoses iotifv emciocrnotbroial,l wmheitlaebsoulpispmle.m Eexnptoastiuorne wofit hPA
thbeiloifpiolmpes pttiod eliaqtuaidv eAry lroewduccoendc enbitorafitlimon (m1emtagb/oLli)smca utsoe dcfau. rt1h5e%r reodfu cthtieo npionsbitaivctee rciaolnmtreotla, bwolhicile
acstuivpitpyle-mtoen5%tatoiofnp owsiittihve tchoen trlioplo.peptide at a very low concentration (1 mg/L) caused further
redLuicqtuioind iBn baapcptleireidal amloentaebcoaliucs aecdtivointyly - ptoa r5t%ia lorfe pdouscittiivoen coofntbraoclt. erial metabolic activity for the
majoritLyiqoufisdtr aBi nasp.pItlsiesdu paplolenme ecnatuastieodn owniltyh tphaertliiaplo preedputicdtieoant aofc obnaccetenrtiraalt imonetoafb4o–l8icm agct/ivLitryed fuocre dthe
thmeabjioorfiitlym osfo sftrSaEin,sS.A It,sE sCup,apnledmEeFntbaytioant lweaitsht 9th0e% l.ipTohpeepPAtidbei oafit alm cowncaesnntroattaioffne cotfe 4d–8b ymlgiq/Lu irdedBuacted
allt.hIet sbcioofmilmbins aotfi oSnEw, SiAth, tEhCe,l iapnodp eEpFt ibdye aatt cleoanscte 9n0t%ra.t iTohnes oPfA1 2b8ioafnildm6 w4masg n/oLt raefdfeuccteedd tbhye lbiqauctiedr iBa lat
maeltla. bIotsl icsommtboincaa.ti4onan wdi2th0 %th,er elisppoepcteipvteildy.e at concentrations of 128 and 64 mg/L reduced the bacterial
meTtahbeolliispmop toep ctai.d 4e aanpdp 2li0e%d, arleosnpeecwtivaeslyh.i ghly active against biofilms formed by SA and SE at
concenTtrhaet iolinpsopoefp3ti2dea nadpp1l6iedm ga/loLn,er ewspase chtiivgehllyy. aActpivpel icaagtaiionnsto bfiothfielmsas mfoermcoendc ebnyt rSatAio nanodf SthEe at
lipcoopnecpentitdraetrioednsu coefd 3m2e taanbdo li1c6a cmtigv/iLty, irnesthpeecEtiFvbeliyo.fi lAmpbpyliccaat.io95n% o.fG trhaem s-namegea ticvoencsetrnatirnasticounl tuorfe dthe
onlippoolpyesptytirdeen eresduurfcaecde smtuetranbeodlioc uatcttoivbitey minu tchhel EesFs bsieonfsilimtiv beyto cath. e95li%p.o Gperpamtid-ne,ewgahtiicvhe csatruasiends cpualrttuiarled
reodnu cptioolnysotyfrbeancet esruiarlfamceesta tbuorlnisemd .oFuotr tPoA b,ew meuobchse rlevsesd smenesditiiuvme taon ttihbea cltieproiapleapcttidivei,t ywohniclyh actatuhseed
hipgahretsitalc orendceuncttrioanti oonf— ba1c2t8ermiagl /mLe.tabolism. For PA, we observed medium antibacterial activity only
at the highest concentration—128 mg/L.
Int. J. Mol. Sci. 2019, 20, x 11 of 20
Int.J.Mol.Sci.2019,20,393 10of19

Figure7.ActivityofCLliquidsaloneandsupplementedwiththelipopeptideappliedatconcentrations
Figure 7. Activity of CL liquids alone and supplemented with the lipopeptide applied at
of 1–128 mg/L against biofilms formed on polystyrene plates by: (A) SE; (B) SA; (C) EF; (D) EC;
concentrations of 1–128 mg/L against biofilms formed on polystyrene plates by: (A) SE; (B) SA; (C)
and(E)PA.Theresultsarepresentedasthepercentageofmetabolicactivityincomparisontopositive
EF; (D) EC; and (E) PA. The results are presented as the percentage of metabolic activity in
(100%)andnegative(0%)controls;RSD≤15%.
comparison to positive (100%) and negative (0%) controls; RSD ≤ 15%.
2.3.2. BiofilmsFormedonCLs
2.3.2. Biofilms Formed on CLs
As described in Section 2.2, the antibacterial effect of CL solutions as well as the lipopeptide
applieAds adleosnceribdeode sinn oStecltaiostn o2n.2c,e ththee aangtiebnatcstearrieal reefpfelacct eodf wCLit hsoMluHtioBnsII .asS uwpeplll eams ethneta ltiipoonpoefptCidLe
saopluptliioedn sawlointhe tdhoeesl ipnoopt elpastitd oenscieg nthifiec aangtelnytsim aprer orveepdlatcheedi rwainthti bMioHfilBm IIa. cStiuvpitpyl,ewmheincthatrioenm aoifn eCdL
asfotelurttihoensw withitdhr athwea lliopfoapcetpivtiedaeg seingtnsi(fiFciagnutrley 8i)m. pTrhoevseydn ethrgeiisr tiacnetifbfeioctfiwlma saecstpiveictyia, llwyhniochti creeamblaeinfoerd
PaAft,eEr Cthaen wdiEthF.dWrahweanl tohfe aCctLivseo laugteionntss (oFrigliuproep e8p).t iTdheew syerneeargpipsltiiecd efafleocnt ewaagsa iensspteEcCialalyn dnPoAticbeiaobfilelm fos,r
tPhAem, EeCta baonldic EacFt.i vWitiheesno fthbea cCteLri asowluetrieonpsa rotira llliypooprefupltliyder ewstoerree da.pSpulpiepdl eamloennet aatigoaninosft bEoCth alinqdu iPdAs
wbiitohfitlhmesl,i ptohpe empteidtaebaotlaicc oanctcievnittireast ioonf obfa4ctmergia/ Lwreerdeu pceadrtbiaalclyte roiar lfmuleltya broelsitsomretdo. cSau.2p%pl.eAmpenertmatiaonne notf
abnodths ulifqfiuciiednst wacittihv itthyea lgiapionpsetpPtAidwe aast oab ctaoinnceednatrfatetirotnh eoaf p4p mlicga/tLio rnedofulcieqdu ibdaBctweriitahl tmheetlaipboopliespmt idtoe caat.
a2c%o.n Ace npterramtiaonneonft 3a2nmd gs/ufLf.icTioennte aacrtliyvtitoyt aallgyairnesmt oPvAe (wmaest aobbotaliicnaedct iavfitteier sthcae. a1p–p2l%ic)atthioenE oFf bliioqfiulimd sB
fwroimth tthhee sluiprofapceepotifdCe Last, al icqounidcesnAtraatniodnB owf 3e2re msug/pLp. lTemo neneaterdlyw toittahlltyh erelmipoovpee p(mtideetaabtocliocn accetnivtritaiteiso ncas.
o1f–126%a) ntdhe3 2EFm gb/ioLf,ilrmessp fercotmiv etlhy.e SsAurwfacaes poef rCmLasn, elniqtluyidelsi mAi naantedd Bf rowmerCe Lssupopnlceemthenetleidp owpeitpht itdhee
wliapsopadepdteiddea attc oconncceenntrtaratitoionnsso off8 1a6n adnd4 3m2g m/Lg/tLo, rlieqsupiedctAivealnyd. SBA, rwesapse pcetirvmelayn.eDntelsyp eitleimthineaftaecdt ftrhoamt
SCELbsi oofinlcme stwhee relipsuopsceepptitdibel ewtoast haedadgeedn tastw choennceanptpraliteiodnasl oonfe ,8w aenadl so4 nmogti/cLe dtoa slyiqnueirdg isAti caenfdfe cBt,
breetswpeecetnivtehleyl. iDpoepspeipttei dtheea fnadctC thLasto SluEt bioionfsi.lms were susceptible to the agents when applied alone, we
also noticed a synergistic effect between the lipopeptide and CL solutions.