molecules Article Antimicrobial Activity and Stability of Short and Long Based Arachnid Synthetic Peptides in the Presence of Commercial Antibiotics IvanArenas1,ElbaVillegas2,OliverWalls3,HumbertoBarrios4,RamonRodríguez3and GerardoCorzo1,* 1 DepartamentodeMedicinaMolecularyBioprocesos,InstitutodeBiotecnología, UniversidadNacionalAutónomadeMéxico,UNAM.ApartadoPostal510-3,Cuernavaca,Morelos62250, Mexico;arsoivan@gmail.com 2 CentrodeInvestigaciónenBiotecnología,UniversidadAutónomadelEstadodeMorelos, Av.Universidad2001,Cuernavaca,Morelos62210,Mexico;elbav@uaem.mx 3 LaboratoriosLiomontSAdeCV,AdolfoLópezMateos68,Cuajimalpa,CuajimalpadeMorelos, MéxicoCity05000,Mexico;owalls@liomont.com.mx(O.W.);rrodrigu@liomont.com.mx(R.R.) 4 InstitutodeSaludPública,Av.Universidad655,SantaMaríaAhuacatitlán,Cuernavaca,Morelos62100, Mexico;humberto.barrios@insp.mx * Correspondence:corzo@ibt.unam.mx;Tel.:+52-777-317-1209;Fax:+52-777-317-2388 AcademicEditor:PeterJ.Rutledge Received:20November2015;Accepted:3February2016;Published:17February2016 Abstract:Fourantimicrobialpeptides(AMPs)namedPin2[G],Pin2[14],P18KandFA1werechemically synthesized and purified. The four peptides were evaluated in the presence of eight commercial antibioticsagainstfourmicroorganismsofmedicalimportance:Escherichiacoli,Staphylococcusaureus, Pseudomonasaeruginosa,andKlebsiellapneumoniae. Thecommercialantibioticsusedwereamoxicillin, azithromycin,ceftriaxone,gentamicin,levofloxacin,sulfamethoxazole,trimethoprimandvancomycin. ThebestAMPagainstP.aeruginosawasthepeptideFA1,andthebestAMPagainstS.aureuswas Pin2[G]. Both FA1 and Pin2[G] were efficient against E. coli, but they were not effective against K.pneumoniae. AsK.pneumoniaewasresistanttomostofthecommercialantibiotics,combinationsof theAMPsFA1andPin2[G]werepreparedwiththeseantibiotics.Accordingtothefractionalinhibitory concentration (FIC) index, the best antimicrobial combinations were obtained with concomitant applicationsofmixturesofFA1withlevofloxacinandsulfamethoxazole. However,combinationsof FA1orPin2[G]withotherantibioticsshowedthattotalinhibitoryeffectofthecombinationswere greaterthanthesumoftheindividualeffectsofeithertheantimicrobialpeptideortheantibiotic. WealsoevaluatedthestabilityoftheAMPs. TheAMPPin2[G]manifestedthebestperformance insalinebuffer,insupernatantsofbacterialgrowthandinhumanbloodplasma. Nevertheless,all AMPswerecleavedusingendoproteolyticenzymes. Thesedatashowadvantagesanddisadvantages of AMPs for potential clinical treatments of bacterial infections, using them in conjunction with commercialantibiotics. Keywords: antibiotic;antimicrobialpeptide;peptide;bacteria 1. Introduction Sincethedevelopmentofpenicillininthe1940s,thesynthesisanduseofdifferentantibioticshas hadanimportantimpactonhumanhealth;however,theemergenceofresistantbacteriahasmade bacterialinfectionsincreasinglydifficulttotreatwithavailableantibiotics. Thus, oneofthemajor challengestotoday’sworldhealthsystemsistheappearanceandspreadofresistancetoantibiotics. Molecules2016,21,225;doi:10.3390/molecules21020225 www.mdpi.com/journal/molecules Molecules2016,21,225 2of17 Many pathogenic bacteria that were once sensitive to conventional antibiotics are now multidrug Molecules 2016, 21, 225 2 of 18 resistant,reducingthetherapeuticvalueoftheseantibiotics[1]. Thishasencouragedasearchforleads indriecsaistitnangtn, roevdeulcainngti bthioe ttihcseraampeountgic nvaatluurea olfc tohmespeo aunntidbsiotthicast [a1r]e. Tahbilse htaosb eynpcaosusrathgeedk an osewanrcmh feocrh laenaidssm s forinddriucagtirnegs insotavnelc ean[t2ib].ioTtihcse aamnotinmg incarotubriaall cpoempptoiduensds(A thMatP asr)e mabalye tcoo bnysptiatsust tehae nknaoltwenrn mateicvheanfoisrmths is, serfvoirn gdrduigre cretlsyisotarnicne c[o2m]. bTihnea taionntimwiictrhocbliaasl spiceapltaidnetsib (iAotMicsPs[)3 ,m4]a.yH coownesvtietur,tetw aon carlitteicrnalatdivisea dfovra nthtaisg, es ofstherevsienga ndtiirmecitclyro obri ainl pcoemptbiidneastioanre wthitehi rclsaiszseicaaln adnttihbeioirticpso [o3r,4s].t aHboilwiteyvienr, tthweop crreitsiceanlc deisoafdpvraontteaogleyst ic of these antimicrobial peptides are their size and their poor stability in the presence of proteolytic enzymes. Therefore,theaimofthisworkwastotestfourdesignedsyntheticpeptides,basedonnative enzymes. Therefore, the aim of this work was to test four designed synthetic peptides, based on native arachnidpeptidesofdifferentsizes,andtoobservetheirstabilityunderdifferentconditionsaswellas arachnid peptides of different sizes, and to observe their stability under different conditions as well theirantimicrobialcapacityintheabsenceandpresenceofexistingantibiotics. as their antimicrobial capacity in the absence and presence of existing antibiotics. 2. Results 2. Results 2.1. IsolationandSequenceAnalysis 2.1. Isolation and Sequence Analysis FourpeptideswerechemicallysynthesizedandpurifiedbyreversephaseHPLC(Figure1).Theretention Four peptides were chemically synthesized and purified by reverse phase HPLC (Figure 1). The timeofeachpeptide,underthesameconditions,showedthattheirhydrophobiccharacterranged retention time of each peptide, under the same conditions, showed that their hydrophobic character fromlesstomorehydrophobic;Pin2[14]50, >50, >50 and >50 µg/mL, for P18K were >50, >50, >50 and >50 µg/mL, for FA1 were 12.5, 25, >50 and >50 µg/mL and for Pin2[G] were 50, >50, 25 and >50 µg/mL for E. coli, P. aeruginosa, S. aureus and K. pneumoniae, respectively (Figure 2 and Table 2). Consequently, no MICs were found for Pin2[14] and P18K in the case of all four microorganisms tested. The most resistant bacteria was K. pneumoniae and relatively the most vulnerable was E. coli. Molecules2016,21,225 4of17 Althoughtherewasnoclearcorrelationbetweenthemolecularsize,hydrophobicityandbiological activityinthecaseofallfourpeptidestested,thelargestandmosthydrophobicpeptideswerethe mosteffectiveagainstallfourbacteria,asopposedtotheshortestandleasthydrophobicpeptides. ThismeansthatthelargestpeptidesFA1andPin2[G]manifestedthemosteffectiveantimicrobial activitytowardsallfourbacteriatested,however,thepeptideFA1wasthemostactiveagainstGram negativebacteria,andPin2[G]wasthemostpotentagainsttheGrampositiveS.aureus. Bacteria-free wellabsorbancesareindicatedbyadottedlineinFigure2;thus,theminimalinhibitoryconcentrations (MICs)forPin2[14]were>50,>50,>50and>50µg/mL,forP18Kwere>50,>50,>50and>50µg/mL, forFA1were12.5,25,>50and>50µg/mLandforPin2[G]were50,>50,25and>50µg/mLforE.coli, P.aeruginosa,S.aureusandK.pneumoniae,respectively(Figure2andTable2). Consequently,noMICs werefoundforPin2[14]andP18Kinthecaseofallfourmicroorganismstested. Themostresistant bacteriawasK.pneumoniaeandrelativelythemostvulnerablewasE.coli. Table2. MinimalinhibitoryconcentrationofpeptidesandantibioticsagainstE.coli, P.aureginosa, S.aureusandK.pneumoniae. MinimalInhibitoryConcentrations(µg/mL)a Antimicrobials E.coli P.aureginosa S.aureus K.pneumoniae Peptides Pin2[14] >50 >50 >50 >50 P18K >50 >50 >50 >50 Pin2[G] 50 >50 25 >50 FA1 6.25 25 >50 >50 Antibiotics Amoxicillin 12.5 >50 1.5 >50 Gentamicin 1.5 1.5 1.5 12.5 Vancomycin >50 >50 1.5 >50 Ceftriaxone 1.5 12.5 1.5 >50 Sulfamethoxazole >50 >50 >50 >50 Levofloxacin 1.5 1.5 1.5 50 Azitromicin 1.5 12.5 3.1 >50 Trimethoprim 1.5 >50 1.5 >50 aMICaccordingtodataonFigures2and3. 2.3. AntimicrobialActivitiesofSelectedAntibiotics Similarly,alleightantibioticswereassayedforantimicrobialactivitytowardsE.coli,S.aureus, P.aeruginosaandK.pneumoniae(Figure3). S.aureuswasthemostsusceptiblebacteriatothecommercial antibioticstested;itonlyshowedresistancetosulfamethoxazole(Figure3A).P.aeruginosawasresistant toamoxicillin,vancomycin,sulfamethoxazoleandtrimethoprim(Figure3B).E.coliwasthesecond most susceptible bacteria, with resistance to vancomycin and to some extent to sulfamethoxazole (Figure 3C). The most resistant bacteria were K. pneumoniae, which could grow in the presence oftrimethoprim, sulfamethoxazole, azithromycin, amoxicillin, ceftriaxoneandvancomycin(upto 50µg/mL)andtosomeextentinthepresenceoflevofloxacin(upto25µg/mL);however,K.pneumoniae wassusceptibletogentamicinattheconcentrationsused(Figure3D).Table2summarizestheMIC foundforallfourbacteria. Interestingly,allfourbacteriawereresistanttosulfamethoxazole,butall weresusceptibletolevofloxacinandgentamicin(exceptK.pneumoniae). Molecules 2016, 21, 225 5 of 18 Table 2. Minimal inhibitory concentration of peptides and antibiotics against E. coli, P. aureginosa, S. aureus and K. pneumoniae. Minimal Inhibitory Concentrations (µg/mL) a Antimicrobials E. coli P. aureginosa S. aureus K. pneumoniae Peptides Pin2[14] >50 >50 >50 >50 P18K >50 >50 >50 >50 Pin2[G] 50 >50 25 >50 FA1 6.25 25 >50 >50 Antibiotics Amoxicillin 12.5 >50 1.5 >50 Gentamicin 1.5 1.5 1.5 12.5 Vancomycin >50 >50 1.5 >50 Ceftriaxone 1.5 12.5 1.5 >50 Sulfamethoxazole >50 >50 >50 >50 Levofloxacin 1.5 1.5 1.5 50 Azitromicin 1.5 12.5 3.1 >50 Trimethoprim 1.5 >50 1.5 >50 a MIC according to data on Figures 2 and 3. 2.3. Antimicrobial Activities of Selected Antibiotics Similarly, all eight antibiotics were assayed for antimicrobial activity towards E. coli, S. aureus, P. aeruginosa and K. pneumoniae (Figure 3). S. aureus was the most susceptible bacteria to the commercial antibiotics tested; it only showed resistance to sulfamethoxazole (Figure 3A). P. aeruginosa was resistant to amoxicillin, vancomycin, sulfamethoxazole and trimethoprim (Figure 3B). E. coli was the second most susceptible bacteria, with resistance to vancomycin and to some extent to sulfamethoxazole (Figure 3C). The most resistant bacteria were K. pneumoniae, which could grow in the presence of trimethoprim, sulfamethoxazole, azithromycin, amoxicillin, ceftriaxone and vancomycin (up to 50 µg/mL) and to some extent in the presence of levofloxacin (up to 25 µg/mL); however, K. pneumoniae was susceptible to gentamicin at the concentrations used (Figure 3D). Table 2 summarizes the MIC Molecufleosu2n01d6 ,fo21r ,a2l2l 5four bacteria. Interestingly, all four bacteria were resistant to sulfamethoxazole, but al5l of17 were susceptible to levofloxacin and gentamicin (except K. pneumoniae). Molecules 2016, 21, 225 6 of 18 Figure 3. Cont. Figure 3. Dose-response activities of selected antibiotics against bacteria. The eight commercial Figure3.Dose-responseactivitiesofselectedantibioticsagainstbacteria.Theeightcommercialantibiotics antibiotics were tested against the growth of (A) Staphylococcus aureus ATCC 25923; (B) Pseudomonas weretestedagainstthegrowthof(A)StaphylococcusaureusATCC25923; (B)Pseudomonasaeruginosa aeruginosa ATCC 27853; (C) Escherichia coli ATCC 25922; and (D) Klebsiella pneumoniae ST258 in ATCC27853;(C)EscherichiacoliATCC25922;and(D)KlebsiellapneumoniaeST258inMuller-Hinton Muller-Hinton broth. Bacteria-free well absorbances are indicated by a dotted line. broth.Bacteria-freewellabsorbancesareindicatedbyadottedline. 2.4. Biological Activities of Selected Peptides and Antibiotics 2.4. BiologicalActivitiesofSelectedPeptidesandAntibiotics As K. pneumoniae was the most resistant bacteria to the AMPs and showed significant resistance Atos mKo.sptn oefu tmheo naniateibwioatiscst,h ceommboisntarteiosnisst oafn tthbea bcetsetr AiaMtoPst;h FeAA1 ManPds Painnd2[Gsh],o wweerde csoigmnbiifinceadn wtirtehs aisllt ance eight antibiotics and tested against K. pneumoniae (Figures 4 and 5). Figure 4 shows the antimicrobial tomostoftheantibiotics,combinationsofthebestAMPs;FA1andPin2[G],werecombinedwithall activity of Pin2[G] in combination with the eight antibiotics. As observed in Figure 3D, K. pneumoniae eightantibioticsandtestedagainstK.pneumoniae(Figures4and5). Figure4showstheantimicrobial was susceptible to levofloxacin and gentamicin; therefore, as expected the mixtures of the AMPs with activityofPin2[G]incombinationwiththeeightantibiotics. AsobservedinFigure3D,K.pneumoniae these two antibiotics showed the best results. Furthermore, combinations of FA1 with trimethoprim, wassusceptibletolevofloxacinandgentamicin;therefore,asexpectedthemixturesoftheAMPswith sulfamethoxazole, azithromycin, ceftriaxone and vancomycin improved the inhibition of K. pneumoniae thesettow soomaen teixbtieontti c(Fsigsuhroew 4)e. dLitkheewbisees, tthree scuolmtsb.inFautriothnse romf Poirne2,[Gco] mwibthin sautlifoanmsetohfoFxAaz1olwe, iatzhitthrriommeytchion,p rim, sulfamceefttrhioaxxoanzeo laen,da zvitahnrcoommyycciinn ,scoemfterwiahxaotn iemapnrdovveadn cthoem iynhciinbiitmionp roofv tehdist hbaecitnehriiab. iHtioonwoevfeKr,. phnigehuemr oniae tosomcoenecxentetrnatti(oFnigs uorfe P4i)n.2L[Gik]e wweirsee ,rtehqeuicroemd, biinn aotridoenrs toof oPbisne2r[vGe ]gwreiathters uinlfhaimbietitohno xefafzecotlse ,(Faizgiuthrer o5m). ycin, ceftriaTxabolne e3 asunmdmvaarnizceosm thyec binesst ocomnecewnhtraattioimnsp foror vinehdibtihtiengi nthhei bgirtoiwontho off tKh. ipsnbeuamctoenriiaae.. AHlsoow, tehve esra,mhei gher concetnatbrlaet isohnoswos ftPhein F2I[CG i]nwdeexreesr, eiqnudiicraetdin,gin thoerd seyrnteorgoyb, saedrdvietigvree eaftfeerctisn ohri baintitoagnoenfifsemct sof( Fthigeu mreix5t)u.rTesa ble3 used [9]. The best combinations of the peptide FA1 were with levofloxacin, and sulfamethoxazole, summarizesthebestconcentrationsforinhibitingthegrowthofK.pneumoniae. Also,thesametable showing FIC indexes lower than 0.5. Furthermore, combinations of the peptide FA1 with azithromycin, showstheFICindexes,indicatingthesynergy,additiveeffectsorantagonismofthemixturesused[9]. ceftriaxone and vancomycin also demonstrated the inhibition of K. pneumoniae showing that the total ThebestcombinationsofthepeptideFA1werewithlevofloxacin,andsulfamethoxazole,showingFIC effect of such mixtures was greater than the sum of the individual effects (here, FIC <1). Concerning indexeslowerthan0.5. Furthermore,combinationsofthepeptideFA1withazithromycin,ceftriaxone the performance of the peptide Pin2[G] with antibiotics against K. pneumoniae, the best combinations andvoafn tchoem pyecpitnidael swoedree mwoitnhs tarzaitthedrotmhyeciinnh ainbdit iloenvooffloKx.apcinne uemveonn itaheosuhgohw thineg FtIhCa itntdheexteos twaleerfef eloctwoefr such mixtutrheasnw 1 ainsdgirceaatitnegr tahna andtdhiteivseu emffeocft trhaethienrd tihvaind ua aslyenfefregcitcs o(nhee raec,cFoIrCdin50 >50 >50 >50 2> FA1-Azithromycin 25 1.2 >50 >50 <0.524 FA1-Ceftriaxone 25 1.2 >50 >50 <0.524 FA1-Gentamicin 50 3.1 >50 12.5 <1.25 FA1-Levofloxacin 12.5 12.5 >50 50 <0.48 FA1-Sulfamethoxazole 6.25 1.2 >50 >50 <0.15 FA1-Trimethoprim 50 3.1 >50 >50 <1.062 FA1-Vancomycin 12.5 25 >50 >50 <0.75 Pin2[G]-Amoxicillin >50 >50 >50 >50 2> Pin2[G]-Azithromycin 25 25 >50 >50 <1 Pin2[G]-Ceftriaxone 50 12.5 >50 >50 <1.25 Pin2[G]-Gentamicin 6.25 12.5 >50 12.5 <1.25 Pin2[G]-Levofloxacin 25 25 >50 50 <1 Pin2[G]-Sulfamethoxazole 50 1.2 >50 >50 <1.024 Pin2[G]-Trimethoprim >50 >50 >50 >50 2> Pin2[G]-Vancomycin 50 50 >50 >50 <2 aThebestconcentrationofmixturesarebasedontheresultsshowninFigures4and5;b,cTheMICsarebased ontheresultsshowninFigures2and3;dFICIndex;<0.5synergistic;>0.5and4antagonistic. 2.5. StabilityofPeptidesinSalineBuffer,HumanBloodPlasmaandinthePresenceofEnzymes Oneoftheproblemscitedforusingantimicrobialpeptidesreferstotheirstabilityindifferent fluidssuchasaqueoussolutions,orinthepresenceofhumanbloodplasmaand/orinthepresenceof proteolyticenzymes. Figure6showsthatthepeptidesmaybestableinabufferedsalinesolutionorin asterileexcipientabletocarrythesepeptidesasactiveingredients.Asobserved,thepeptideFA1isless stableinsalinesolutionduringthetimeofexperimentation. ThedecrementinconcentrationofFA1in thesalinesolutionmaybeduetoabsorptionontothevialsurface. Although,thepeptideFA1isless hydrophobicthanthepeptidePin2[G],FA1maypresentlargerhydrophobicclustersthatinteractwith thevialsurface. Figures7–10showthestabilityoftheAMPsinhumanbloodplasmaandinbacterial supernatantsforaperiodof12hat37˝C.Firstly,Pin2[G]wasthepeptidethatmaintainedmostof itsintegrityundertheseconditions. Onequarterofitsconcentrationwasfoundinhumanplasma (Figure7A,B),anditwasnotcleavedbythesupernatantsofS.aureus(Figure7C)andP.aeruginosa (Figure7D)afteranincubationperiodof12hat37˝C.Secondly,Pin2[14]wasnotfoundafterextraction from human plasma (Figure 8A,B), and was not cleaved by supernatants of S. aureus (Figure 8C); neitherwasitfoundaftertreatmentwithP.aeruginosasupernatants(Figure8D).Thirdly,neitherwas P18Kobtainedafterextractionfromhumanplasma(Figure9A,B),asitwasprobablysequesteredor cleavedbyproteinsinthesupernatantsofS.aureus(Figure9C)andofP.aeruginosa(Figure9D).Finally, FA1wasnotfoundafterextractionfromhumanplasma(Figure10A,B)andonlyasmallamountof FA1wasobtainedaftertreatmentwithsupernatantsfromS.aureus(Figure10C),butwasnotfound aftertreatmentwithsupernatantsofP.aeruginosa(Figure10D).Likewise,notablynoneoftheAMPs, suchasPin2[14],P18KandFA1,wererecoveredafterinteractionwithhumanplasma,evenafterthe firstfewminutesofdilution,suggestingthatthesethreepeptideswerestronglyboundtoproteins fromhumanplasma. Molecules2016,21,225 9of17 Molecules 2016, 21, 225 10 of 18 Molecules 2016, 21, 225 10 of 18 Figure 6. Stability of peptides under saline buffer. The AMPs were diluted in PBS (pH 7.4) and FFiigguurree 66.. SStatabbiliiltiytyo fopf eppetipdteisduesn duenrdsearli nsealbinueff ebru.Tffheer. ATMheP sAwMerPesd wiluetreed diniluPtBeSd( pinH P7B.4S) a(npdHi n7c.u4)b aatnedd iainn3cc7uub˝baCatteefdodr aas e33v77 e °°nCCd ffaooyrr s ss.eevveenn ddaayyss.. Figure 7. Stability of Pin2[G] in human blood plasma and bacterial supernatants. The peptide was FFiigguurree 77.. SSttaabbiilliittyy ooff PPiinn22[[GG]] iinn hhuummaann bblloooodd ppllaassmmaa aanndd bbaacctteerriiaall ssuuppeerrnnaattaannttss.. TThhee ppeeppttiiddee wwaass either diluted in human blood or bacterial supernatants, incubated at 37 °C for 12 h and then extracted. eeiitthheerr ddiilluutteedd iinn hhuummaann bblloooodd oorr bbaacctteerriiaall ssuuppeerrnnaattaannttss,, iinnccuubbaatteedd aatt 3377 °˝CC ffoorr 1122 hh aanndd tthheenn eexxttrraacctteedd.. (A) Pin2[G] was diluted with blood plasma, immediately extracted and separated by HPLC (control); ((AA)) PPiinn22[[GG]] wwaass ddiilluutteedd wwiitthh bblloooodd ppllaassmmaa,, iimmmmeeddiiaatteellyy eexxttrraacctteedd aanndd sseeppaarraatteedd bbyy HHPPLLCC ((ccoonnttrrooll));; (B) Pin2[G] was diluted with blood, incubated for 12 h at 37 °C, extracted and separated by HPLC; ((BB)) PPiinn22[[GG]] wwaass ddiilluutteedd wwiitthh bblloooodd,, iinnccuubbaatteedd ffoorr 1122 hh aatt 3377 ˝°CC,, eexxttrraacctteedd aanndd sseeppaarraatteedd bbyy HHPPLLCC;; (C) Pin2[G] was diluted with Staphylococcus aureus supernatant, incubated for 12 h at 37 °C, extracted ((CC)) PPiinn22[[GG]]w waassd diliulutetdedw witihthS tSatpahpyhloycloocccouccsuasu areuurseusus psuerpneartnaantta,nint,c uinbcautebdatfeodr 1fo2rh 1a2t h3 7at˝ C37, e°xCt,r aecxtterdacatnedd and separated by HPLC; and (D) Pin2[G] was diluted with Pseudomonas aeruginosa supernatant, saenpda rsaetepdarbayteHdP bLyC ;HanPdLC(D; )aPnidn 2([DG)] wPians2d[Gilu] tewdaws idthiluPtseeudd owmiothn aPssaeeurudgoimnoosnaassu apeerrungaintaonsta, isnucpuebranteadtafnotr, incubated for 12 h at 37 °C, extracted and separated by HPLC. The arrows show the presence 1in2chubatat3e7d˝ Cfo,re x1t2ra cht eadt a3n7d °sCep, aerxattreadctbeyd HaPnLdC s.eTphaeraatrerdow bsys hHoPwLtCh.e Tphrees eanrcroewofs Psihno2[wG ]t.he presence of Pin2[G]. of Pin2[G]. Molecules2016,21,225 10of17 Molecules 2016, 21, 225 11 of 18 Molecules 2016, 21, 225 11 of 18 Figure 8. Stability of Pin14[G] in human blood plasma and bacterial supernatants. The peptide was Figure8.StabilityofPin14[G]inhumanbloodplasmaandbacterialsupernatants.Thepeptidewas Figure 8. Stability of Pin14[G] in human blood plasma and bacterial supernatants. The peptide was either diluted in human blood or bacterial supernatants, incubated at 37 °C for 12 h and then eitherdilutedinhumanbloodorbacterialsupernatants,incubatedat37˝Cfor12handthenextracted. either diluted in human blood or bacterial supernatants, incubated at 37 °C for 12 h and then extracted. (A) Pin14[G] was separated by HPLC (control); (B) Pin14[G] was diluted with blood, (A)Pin14[G]wasseparatedbyHPLC(control);(B)Pin14[G]wasdilutedwithblood,incubatedfor extracted. (A) Pin14[G] was separated by HPLC (control); (B) Pin14[G] was diluted with blood, 1in2cihuncabutabt3ea7dte˝ dCfo ,freo xr1 t2r1 a2hc t hea dta ta3 n73d 7° sC°eC,p , eaexrxatrttraeacdcttebeddy HaannPddL Cssee;pp(Caarr)aaPtteeidnd 1 b4by[yG H]HPwPLaLCsC;d ; (iCl(uC) t)eP dPiniw1n41i[t4Gh[G]S t]wa pwahsa ysdlo iclduoictleucdute sdwa uiwtrhei uths sSutapSpetharypnhlaoytclaooncccotuc,csiun ascu auruebruaesut essd usupfoperrenr1na2atathannta,tt ,i ni3n7ccuu˝bCbaa,tteeedxd t ffrooarrc t11e22d hha aantt d 3377s e °°CpCa, ,er eaxxttretradacctbetyedd Ha naPndLd sC esp;eaaprnaadrtae(tdDe db)y bP Hyin PH1L4PC[LG; Ca]n; wdan ads d(Dilu()D tPe)d inPw1in4i1[thG4[]PG sw]e uwadsao smd idolnuilautestdead erw uwigtiihtnh o PsPasseseuuuddpooemmrononnaaatsas naatee,rriuunggciiunnobossaaat essduupfpoeerrrn1na2atathannat,tt ,i3 ni7ncu˝cCubab,teaextdet rdfa ocfrto er1d 21 a2hn dhat s ae3tp7 a 3°r7Ca t,°e Cd, extracted and separated by HPLC. The arrows show the presence of Pin14[G]. byeHxtPrLacCte.dT haneda rsreopwarsastehdo wbyt hHePpLrCe.s Tenhcee arorfoPwins 1s4h[oGw] .the presence of Pin14[G]. FFFiiiggguuurrreee 999... CCCooonnnttt...

Antimicrobial Activity and Stability of Short and Long Based Arachnid Synthetic Peptides in the

Similar Antimicrobial Activity and Stability of Short and Long Based Arachnid Synthetic Peptides in the