h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Medical
Microbiology
Membrane
permeabilization
of
colistin
toward
pan-drug
resistant
Gram-negative
isolates
夽
Yasmine
Fathy
Mohamed
∗,
Hamida
Moustafa
Abou-Shleib,
Amal
Mohamed
Khalil,
Nadia
Mohamed
El-Guink,
Moustafa
Ahmed
El-Nakeeb
PharmaceuticalMicrobiologyDepartment,FacultyofPharmacy,AlexandriaUniversity,Egypt
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t
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c
l
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Articlehistory:
Received17November2013 Accepted7October2014 Availableonline2March2016 AssociateEditor:RoxaneMaria FontesPiazza Keywords: Pan-drugresistant Leakage Permeabilityalteration Colistin Ultrastructure
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b
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Pan-drugresistantGram-negative bacteria,beingresistanttomostavailableantibiotics, representahugethreattothemedicalcommunity.Colistinisconsideredthelast thera-peuticoptionforpatientsinhospitalsettings.Thus,wewereconcernedinthisstudyto demonstratethemembranepermeabilizingactivityofcolistinfocusingoninvestigatingits efficiencytowardthosepan-drugresistantisolateswhichrepresentacriticalsituation.We determinedthekillingdynamicsofcolistinagainstpan-drugresistantisolates.The perme-abilityalterationwasconfirmedbydifferenttechniquesas:leakage,electronmicroscopy andconstructionofanartificialmembranemodel;liposomes.Moreover,selectivityof col-istinagainstmicrobialcellswasalsoelucidated.Colistinwasprovedtoberapidbactericidal againstpan-drugresistantisolates.Itinteractswiththeouterbacterialmembraneleading todeformationofitsoutline,poreformation,leakageofinternalcontents,celllysisand finallydeath.Furthermore,variationsinmembranecompositionofeukaryoticand micro-bialcellsprovideakeyforcolistinselectivitytowardbacterialcells.Colistinselectivelyalters membranepermeabilityofpan-drugresistantisolateswhichleadstocelllysis.Colistinwas provedtobeanefficientlastlinetreatmentforpan-drugresistantinfectionswhicharehard totreat.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Recently,ithasbeenwitnessedworldwidethatGram-negative bacteriaresistant to manyclasses ofantibiotics represents afearfulsituation towardtheemergence ofafuture medi-caldisaster.1 Thereare2termscommonlydescribingthose
夽TheworkwasconductedinPharmaceuticalMicrobiologyDepartment,FacultyofPharmacy,AlexandriaUniversity,Egypt. ∗ Correspondingauthor.
E-mail:[email protected](Y.F.Mohamed).
superbugs; which are multi-drugresistant (MDR) and pan-drugresistant(PDR).AnisolateisconsideredMDRifit exhib-itedresistancetoward5outofthe7anti-pseudomonalclasses of antimicrobial agents, i.e. anti-pseudomonal penicillins, cephalosporins, carbapenems, monobactams, quinolones, aminoglycosides,andcolistin,whileitisaPDRifitshowed resistance toward all 7 anti-pseudomonal antimicrobial
http://dx.doi.org/10.1016/j.bjm.2016.01.007
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
agents,includingcolistin.2Therewas anotherview consid-eringthatPDRisolateswerethoseresistanttoallantibiotics butonlysusceptibletopolymyxins.3,4 Althoughthereisno apparentdefinitionforthetermPDRthroughoutliterature,it generallydenotesresistanceagainstavarietyofantibiotics excludingpolymyxins.5Suchviewisadoptedinthepresent work.Inthepastfewdecadestherehavebeenatremendous increaseinresistancetocurrentlyavailableantibioticsanda significantdeclineindevelopmentofnewones.6Thisleadsto therevivalofolderagentsaspolymyxins,forthetreatmentof suchPDRinfections.2
Polymyxinsareagroupofpolypeptidecationicantibiotics thatwereisolatedfromBacilluspolymyxainthe1940s.7Since then,polymyxinE(colistin)andpolymyxinBwereextensively usedinclinicalpracticeforGram-negativeorganisms.8,9 How-ever, theywere graduallywithdrawn from the market and abandonedduringthelasttwodecadesduetoclaimedreports oftoxicity.Therefore,duringthattime,therehavebeenlimited studieson theclinical use, pharmacokineticsand pharma-codynamicsofcolistin.10 Emergenceofthe PDRpathogens, necessitatedthere-evaluationofpolymyxintherapies.11
Colistinhasbeenrecentlyconsideredaslastoption treat-mentforpatientswithnosocomialPDRinfections,whichhave becomeanimportantpublichealthissue,owingtoits favor-ablepropertiesofrapidbacterialkilling,anarrowspectrum ofactivity,andslowdevelopmentofresistance.12,13 Colistin interactselectrostaticallywiththeoutermembraneof Gram-negativebacteriaandcompetitivelydisplacesdivalentcations whichstabilizethelipopolysaccharidelayerthusdisrupting themembraneintegrity.Itisthensubsequentlytakenupvia theself-promoteduptakepathway.13 Itisbelievedthat col-istinformscracksintheoutermembranewhichpromotesits uptakeinsidethe celland permitsthepassageofdifferent molecules.14Thus,polymyxinsproduceadisruptivedetergent effect,leadingtoincreasedpermeabilityintheouter mem-brane, leakageof the absorbing cytoplasmic contents, cell lysisandfinallydeath.15Thechemicalcompositionof bacte-rialmembranesbeingrichinphosphatidylethanolamineand negativelychargedlipidsallowssuchelectrostaticattraction withcationicpeptidesincontrasttoeukaryoticcellsinwhich cholesterolisthepredominantcomponentprovidingacluefor theselectivityofactiontowardmicrobialversushostcells.16 Suchasituationpromptedthepresentmicrobiologicalstudy toinvestigatethe membranepermeabilityalterationof col-istinanditbactericidaleffectonPDRGram-negativeclinical isolatesincludingAcinetobacterbaumannii,Pseudomonas aeru-ginosa,KlebsiellapneumoniaeandEscherichiacoli.
Materials
and
methods
Microorganismsandantibiotics
Fourclinicalbacterialisolates,identifiedbyclassical micro-scopic and biochemical procedures,17,18 were used in this study. These are: A. baumannii (A182), P. aeruginosa (P103),
E. coli (E9) and K. pneumoniae (K103). The identified iso-lateswere maintained byfreezing in15% glycerolbroth 19 (OxoidLtd.; Basingostok;Hampshire,England).Colistin sul-phatewasobtainedaspowderfromPharcopharmaceutical
Co.,Egypt.Itwasdissolved inwatertoprepare stock solu-tions.Thesusceptibilityofthetestedisolates,tothedifferent classes ofantibiotics wasdetermined bythe standard disc agar diffusion technique according to Bauer et al.20 with somemodifications.21MICofcolistinwasdeterminedbythe standardbrothdilutiontechnique.22
Bactericidalactivityofcolistinusingtheviablecount
technique
Foreachtestedisolate,twoconcentrationsofcolistin(1/2MIC andMIC)werepreparedinsterilenutrientbroth.Each concen-trationwasinoculatedwithovernightculturetogiveafinal inoculumof106cfu/mL.Acontrolwithoutantibioticwas
pre-paredforeachofthetestedisolates.Thesystemsweremixed wellandincubatedat37◦Cwithshaking.Sampleswere asep-ticallywithdrawnfromeachtesttubeat0,1,3,6and24hand seriallydilutedwithsterilesaline.Then,10Lportionswere droppedontothesurfaceofoverdriednutrientagarplates.The plateswerelefttodryandincubatedinvertedat37◦Cfor24h, the resultingcolonieswerecountedand theoriginalviable countwasdetermined.23
Effectofcolistinonthecytoplasmicmembranebyleakage
technique
Bacterialsuspensionsoftheselectedisolateswerepreparedby streakinganovernightbrothcultureontonutrientagarslants (Oxoid Ltd.; Basingostok; Hampshire, England). The slants wereincubatedat37◦Cfor16–18h.Theresultinggrowthof3 slantswasresuspendedin5mLsterile0.9%salinetoproduce heavy inoculum(O.D600 adjustedto2) and transferredinto
steriletest tubes. Theobtainedbacterialsuspensions were centrifuged at12,000×gfor5min.Theformedpelletswere washedtwicewithsterilesalineandthenwereresuspended in5mLsterilesaline.Aliquotsofthepreparedbacterial sus-pensionofeachisolateweretreatedwith50mg/Lofcolistin. Acontrolwasincludedineachtestcontaininguntreated bac-terial suspension.Both the treatedand untreatedbacterial suspensionswereincubatedat37◦Cfor24h.Afterincubation, the bacterialsuspensionswere centrifugedat12,000×gfor 5min.Theabsorbanceoftheclearsupernatantwasestimated at 260 and 280nm against saline solution using the spec-trophotometer(Thermospectronic,Heliosalpha,NC9423UV A1002E,England).24
Effectofcolistinontheleakageofredbloodcells
Onemilliliteroffreshhumanbloodwascentrifugedat2000×g
for5minandthecellswerewashed4×withsterile0.9%saline discardingthesupernatanteverytime.Thesedimentedred blood cells (RBCs) were resuspended in 1mL buffer (5mM sodiumphosphateand150mMsodiumchloride[pH7.4]).The RBCs suspensioninbufferwasdistributedinsterile eppen-dorf tubes in 25L aliquots and 1mL of colistin solution dissolved inthesame bufferwas addedtoeach eppendorf in concentrations ranging from 0.78mg/L to 100mg/L. The resultingsuspensionswereincubatedatroomtemperaturefor 2h.Thesystemswerethencentrifugedat2000×gfor10min. Thereleaseofhemoglobinwasmonitoredbymeasuringthe
absorbanceofthesupernatantat540nmbyspekol(CarlZeiss, Jena,Germany).Absorbanceofcontroluntreatedcellsinsaline was measured and used asa blank. Total haemolysiswas alsomeasuredbylysingcolistin-untreatedRBCswithdistilled water.25
Effectofcolistinontheultrastructureofbacterialcells
usingthetransmissionelectronmicroscope
Twoclinicalisolateswereusedinthisstudy;asensitiveand aninducedresistantpopulation foreach isolate.The resis-tancewasinducedbyserialpassaginginincreasingcolistin concentrations.0.5mLofovernightbrothculturewas inocu-latedinflaskscontaining50mLsterilenutrientbroth.Flasks wereincubatedintheorbitalshakingincubator(100rpm)at 37◦C till reachingacceptable turbidityfor about 4–5h. The selectedclinicalisolateswere treatedwith100mg/Lof col-istinfor1h.Propercontrolslackingcolistinwereincludedfor eachisolatepopulation.Theobtainedbacterialsuspensions werecentrifugedat6000×gfor5min.Thesupernatantswere discarded.Theobtainedcolistin-treatedandcontrolbacterial pelletswerethenresuspendedin3mLTrump’sfixative solu-tionandfurtherprocessedaspreviouslydescribed.26,27 The sampleswere examinedandphotographed atan accelerat-ingvoltageof80kVusingJoelCX100transmissionelectron microscopeattheElectron MicroscopeUnit,Faculty of Sci-ence,AlexandriaUniversity.
Effectofcolistinonthecytoplasmicmembraneusing
artificialcytoplasmicmembranemodel
Negatively-charged unilamellar cholesterol free liposomes werepreparedbyreverse-phaseevaporationmethod.28 The liposomeswerethentreatedbycolistin(100mg/L)for24hand weremorphologicallyexaminedbythephasecontrast micro-scope(Olympus,CX41RF)usingoil-immersionobjectivelens (100×)foranydamageintheirshapefollowingcolistin treat-ment.
Effectofcolistinontheformationofspheroplasts
OnehundredLofexponentialcellculture,adjustedtohave O.D600of0.05wereinoculatedinto3mLMüller-Hintonbroth
containing 0.3M of sucrose and were mixed well. Then, aliquotsof100Lweredistributedinsterileeppendorftubes. TenLofcolistinwereaddedto3eppendorfsinconcentration of1/2MIC.TenLofceftazidimewereaddedto3other eppen-dorfsinconcentrationof1/2MIC.Acontroleppendorfwas includedcontaining0.9%salineinsteadofantibioticsolution. Theeppendorfswereincubatedintheorbitalshaking incu-batorat37◦C,100×g for90min.Sampleswere thentaken andexamined bythe phasecontrast microscopeusing oil-immersionobjectivelens(100×).
Results
and
discussion
Globally,thereisagrowingthreatfromtheemergenceofMDR andPDRorganismsespeciallyGram-negativebacteria,suchas
P.aeruginosa,A.baumannii,KlebsiellaandEnterobacterspeciesin
Table1–MICofcolistinagainstPDRclinicalisolates.
Isolatecode MIC(mg/L)
A182 1.25
P103 0.625
E9 0.625
K103 1.25
hospitalsettings.29PDRpathogensrepresentafearfulclinical situationwithtremendousimplications.So,thisstudyaimed atinvestigatingthedeleteriouseffectsofcolistinonthosePDR pathogens.Thefourbacterialisolatesusedinthisstudywere confirmedtobepan-drugresistantbytheantibiotic suscep-tibilitytestingbeingresistanttoallantibioticclassesexcept colistin(datanotshown).TheMICofcolistinagainstthetested isolatesrangedfrom0.625to1.25mg/Lasdeterminedbybroth dilutionmethod(Table1).Allthetestedisolatesweresensitive tocolistinaccordingtotheCLSIresistancebreakpointwhich
is4mg/L(Table1).
Thedynamicsofkillingofcolistinwasdeterminedbythe viable counttechnique againstthe tested isolatesusing
½
MIC and MIC ofcolistin. Colistin was shownto bearapid bactericidal agentinaconcentration dependentmannerin all the tested PDR isolates. Rapid and significant declines (>2log)inbacterialsurvivalwereobservedafter3hinallthe testedisolatesat1/2MIClevelreachingalmost6logsatthe MIClevel.Therapidbactericidalactivityofcolistinisrelated toitspermeabilizingactiononthecellmembranefollowing self-promoteduptake.30However,re-growthwasobservedas earlyas6handsubstantialre-growthoccurredat24hinall the testedPDRclinical isolatesat1/2MICwhileonly2 iso-latesshowedre-growthattheMIClevel(Fig.1).Thismight becausedbytheheteroresistantsubpopulationswhichgrow probably ataslower rate than the sensitivesubpopulation andhence,temporaryinhibitioncouldbemistaken.This phe-nomenonisofpotentialriskasitcanleadtotherapyfailureif colistinmonotherapyisused.31
A leakage study was conducted to confirm the mem-brane permeability alteration effect of colistin. The effect of50mg/Lofcolistinonthecytoplasmicmembraneagainst 4representativePDRclinicalisolateswasdetermined spec-trophotometrically. The difference between the values of absorbanceat260and280nmofthesupernatantofboththe treatedcellsandtheuntreatedcellscorrespondingtothenet leakageduetotreatmentisillustratedinFig.2.
Colistinresultedinanetlossof260and280nm-absorbing materials.Theextentofleakagedifferedfromoneorganism toanother.ThemaximumleakagewasobservedfortheP103 isolate.
Thisleakageeffectisduetothedestabilizationof mem-brane integrity arising afterthe electrostatic interaction of colistin with the outer membrane displacing the divalent cationsfromtheLPSlayer.32Thisresultsinanincreaseinthe permeabilityofcellmembrane,leakageofcellcontentsand finallycelldeath.
Furthermore, to demonstrate the selectivity of colistin toward microbial membranes, the effect ofcolistin on the haemolysis of human RBCs was studied. The release of hemoglobin was monitored by measuring the absorbance of the supernatants of the different reaction mixtures at
A
10 8 6 4 2 Log sur viv ors , cfu/mL 0 0 6 12 Time (h) 18 24B
10 8 6 4 2 Log sur viv ors , cfu/mL 0 0 6 12 Time (h) 18 24C
10 8 6 4 2 Log sur viv ors , cfu/mL 0 0 6 12 Time (h) 18 24D
10 8 6 4 2 Log sur viv ors , cfu/mL 0 0 6 12 Time (h) 18 24Fig.1–BactericidalactivityofcolistinagainstPDRisolates.(A)A.baumannii(A182),(B)P.aeruginosa(P103),(C)K.pneumonie
(K103)and(D)E.coli(E9).Filledcirclesrepresentthecontroluntreatedcells,filledsquaresrepresent1/2MICofcolistin,filled trianglesrepresentMICofcolistin.
540nm by the spekol. Then, the per cent of haemolysis ofRBCs was calculated. Itis shown in Fig. 3 that the per cent of RBCs haemolysis increased by increasing colistin concentration.Itproducedabout1.3%haemolysisata con-centrationof12.5mg/Lwhichisnearlymorethantentimes the averageMIC ofthe tested PDRclinical isolates.Atlow concentrations,itproducedinsignificanthaemolysislessthan 1%. However, there was a sharp increase in the per cent of haemolysis at concentrations above 50mg/L which are unachievable in vivo. This confirms the selectivity of col-istintowardthecytoplasmicmembraneofthebacterialcells and denotesits in vivo safety at the normaladministered
2.0 1.5 1.0 0.5 0.0 A182 P103 K103 E9 Isolate code Absorbance
Fig.2–LeakageofabsorbingmaterialsfromPDRclinical isolatesinthepresenceofcolistin.Whitebarsrepresent 260nmabsorbingmaterialsandblackbarsrepresent 280nmabsorbingmaterials.
doses.Thecompositionofbacterialcellsbeingrichinanionic phosphatidylglycerolandcardiolipinlikelyprovidesan impor-tantdeterminantforantimicrobialcationicpeptidestotarget microbialmembranes.33
Anotherevidenceforthedamagingeffectofcolistinonthe cellenvelopeisobtainedbyelectronmicroscopeexamination. Theeffectofcolistinontheultrastructureof2representative isolates (A182 and P103) was studied. The cells (sensitive and induced resistant) weretreated by100mg/Lofcolistin for30min,centrifuged,fixedandpreparedbyencapsulating and cuttingultra thinsections from treated anduntreated bacterialcellsedimentsfollowedbyexaminationofthe pre-paredsection under the transmissionelectronmicroscope. A smoothcontinuouscell walland cytoplasmicmembrane structureswereobservedintheuntreatedsensitivebacterial
80 60 40 20 0 780 1560 3125 6250 12 500 25 000 50 000 100 000 Colistin concentration (mg/L) % Hemolysis
Fig.3–Effectofdifferentconcentrationsofcolistinon haemolysisofRBCs.
Fig.4–UltrastructureofcolistintreatedA.baumanniicells.(A1,2,3):SensitiveA.baumanniiA182cells,(B1,2,3):Resistant (induced)A.baumanniiA182cells,A1andB1representthecontroluntreatedcellsforeachcategory.Magnification:40,000×: A2,A3,B3and50,000×:A1,B1,B2.
strains. However, a slightly wavy cytoplasmic outline was observed for the induced resistant one (Fig. 4). Colistin-treatedcellsshowednumerousprojectionsandporesonthe cellwallwhichwasalmostdissolvedwhereasthe cytoplas-micmembranewaspartiallydamaged.Thecontentsofsome treatedcells appeareddepleted aspart of the cytoplasmic material was released through cracks. Such phenomenon wasmoreapparentwiththesensitiveisolatesthanwiththe correspondingisolateswithinducedcolistinresistance(Fig.5). Moreover,theeffectofcolistinonanartificialcytoplasmic membranemodelwasdeterminedbyexaminationunderthe phasecontrastmicroscope.Theeffectofcolistin(100mg/L)
onthe negatively-chargedunilamellar cholesterolfree lipo-somescomparedtothecontroluntreatedonesisillustratedin
Fig.6.Colistinresultedinanoveralldeformationinthe struc-tureofthe artificialcytoplasmicmembranemodel. Various effectswereobservedonthephospholipidmembraneranging fromroughnessanddistortionoftheoutline,poreformation andcompleteruptureofthemembraneandliberationofthe internalcontents.
Itwasathoughtofinterestwhethercolistinaffectsonlythe outermembraneoritalsoaffectsthecellwall.So,theeffectof colistinonthecellwallwascomparedtothatofceftazidime whichisa-lactamantibioticknowntoactonthecellwall
Fig.5–UltrastructureofcolistintreatedP.aeruginosacells.(A1,2,3):SensitiveP.aeruginosaP103cells,(B1,2,3):Resistant (induced)P.aeruginosaP103cells,A1andB1representthecontroluntreatedcellsforeachcategory.Magnification:30,000×: A3,B1,B2,40,000×:A1,B3and50,000×:A2.
by using the isolate P103. Ceftazidime causes spheroplast formationprior tocell lysis inGram-negative bacteria due toinhibitionoftranspeptidases(Penicillin-BindingProteins) which catalyze the cross linking of peptidoglycan chains. Thus,itdisruptsthesynthesisofpeptidoglycanresultingin the release of autolysinswhich enzymatically degradethe cellwallsformingspheroplast,whichisosmotically-sensitive cell lacking rigidity of the cell wall.34 Sucrose 0.3M acts as a stabilizer for the formed spheroplasts. By using sub-inhibitoryconcentrationsofceftazidimeinthepresentstudy, spheroplastswereformedandviewedbythephasecontrast
microscope(Fig.7).Whencomparingthattocolistin,similar structureswereobtainedat1/2MIClevel.Formationofsuch osmoticallyunstablestructuresindicatesthatcolistinnotonly actsonthecytoplasmicmembranebutalsoonthecellwall. Therefore, it could beassumed thattreatment ofsensitive Gram-negativecells withcolistin inthepresenceof hyper-tonicsolutionmighthaveresultedinthereleaseofautolysins whichdegradedmostofthecellwallleavingspheroplasts sup-portedbythe surrounding highosmotic pressure.35 Hence, it can be elucidated that colistin acts on multiple layers ofGram-negative cells,initiallyonthe outer membraneas
Fig.6–Morphologyofliposomesexaminedunderoil-immersionobjectivelens(totalmagnification1000×).(A)Untreated control,(B)Colistin-treated[additionalopticalzoom:A1andB1(2×),A2andB2(3×)].
Fig.7–SpheroplastsofP103isolateformedat
½
MICofceftazidime(B)andcolistin(C)inhypertonicsolutioncomparedto shapeofcontrolcells(A).demonstratedbytheelectronmicroscopystudyandthenon theinnermembraneasshownbythespheroplastsdata.
Inconclusion,itcanbedemonstratedthatcolistinisstill apromisingdrugforthetreatmentofinfectionsduetoPDR clinicalisolates.Itinducesalterationsinthepermeabilityof the bacterial cytoplasmic membrane, leakage ofthe intra-cellularcontentsandultimatelycelldeath.However,colistin shouldnotbemisusedtoavoidtheglobalproblemof devel-opmentofresistance.Furtherinvivoinvestigationsarestill requiredconcerningthepharmacokinetics, pharmacodynam-icsandtoxicodynamicsofcolistin.
Conflict
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interest
Nonedeclared.
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