Antibacterial Efficacy of Silver Nanoparticles on Endometritis Caused by Prevotella ...

20 Pages·2017·7.78 MB·English

International Journal o f
Molecular Sciences
Article
Antibacterial Efficacy of Silver Nanoparticles on
Endometritis Caused by Prevotella melaninogenica
and Arcanobacterum pyogenes in Dairy Cattle
SangiliyandiGurunathan*,Yun-JungChoiandJin-HoiKim* ID
DepartmentofStemCellandRegenerativeBiotechnology,KonkukUniversity,Seoul05029,Korea;
[email protected]
* Correspondence:[email protected](S.G.);[email protected](J.-H.K.);
Tel.:[phone](S.G.);[phone](J.-H.K.)
(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:15March2018;Accepted:11April2018;Published:16April2018
Abstract: Bovine postpartum diseases remain one of the most significant and highly prevalent
illnesseswithnegativeeffectsontheproductivity,survival,andwelfareofdairycows. Antibiotics
are generally considered beneficial in the treatment of endometritis; however, frequent usage of
eachantibioticdrugisreasonfortheemergenceofmultidrugresistance(MDR)ofthepathogenic
microorganisms,representingamajorimpedimentforthesuccessfuldiagnosisandmanagementof
infectiousdiseasesinbothhumansandanimals. Wesynthesizedsilvernanoparticles(AgNPs)with
anaveragesizeof10nmusingthenovelbiomoleculeapigeninasareducingandstabilizingagent,
andevaluatedtheefficacyoftheAgNPsontheMDRpathogenicbacteriaPrevotellamelaninogenica
andArcanobacteriumpyogenesisolatedfromuterinesecretionsamples. AgNPsinhibitedcellviability
andbiofilmformationinadose-andtime-dependentmanner. Moreover,themetabolictoxicityof
theAgNPswasassessedthroughvariouscellularassays. Themajortoxiceffectofcelldeathwas
causedbyanincreaseinoxidativestress,asevidencedbytheincreasedgenerationofreactiveoxygen
species(ROS),malondialdehyde,proteincarbonylcontent,andnitricoxide. TheformationofROSis
consideredtobetheprimarymechanismofbacterialdeath. Therefore,thebiomolecule-mediated
synthesis of AgNPs shows potential as an alternative antimicrobial therapy for bovine metritis
andendometritis.
Keywords: antimicrobialtherapy;endometritis;multipledrug-resistantbacteria;silvernanoparticles;
oxidativestress
1. Introduction
Metritisandendometritishaveasubstantialinfluenceonbovinehealthandproductivity,with
significant economic impacts to the dairy industry. Several studies have provided evidence that
uterineinfectionsareduetobacterialpathogenesisintheuterus[1–7]. Theuterineinfectionscaused
by pathogenic bacteria lead to inflammation and infertility [3]. Uterine disease has unique and
characteristicfeatures,includingalowerconceptionrate,alongwithincreasedintervalsfromcalving
tothefirstserviceorconception[8]. Endometritisisaninflammatorydisease, whichisassociated
withdelayeduterineinvolutionandpoorreproductiveperformance[9]. Endometritisisfrequently
treatedbyintrauterineinfusionofantibiotics[10]. However,theoverloadingandindiscriminateuseof
antibioticsforthetreatmentofuterineinfectionsoranyothermicrobial-relatedinfectionshasledto
theemergenceofantibiotic-resistantstrains. Indeed,theoverwhelmingusageofantibioticshasled
tomultidrugresistance(MDR),prolongedinfectiontreatment,andincreasedmortalityrisk[11,12].
Furthermore,thisincreasedriskofmicrobialresistanceresultsinlesseffectiveconventionaltreatments.
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Therefore,itisnecessarytoovercomethelimitationsoftheconventionalcontinuoususageofantibiotics
inthedairyindustryandagriculture.
The application of nanoparticles has attracted huge interest in several fields, including
biotechnology, biomedical sciences, and veterinary medicine. Several studies have explored the
possibility of high-level nanotherapy in humans; however, the applications of nanotechnology in
veterinary medicine have not reached the same level, and remain in a relatively innovative stage.
Veryrecently,nanoparticleshavebeenusedasnutraceuticals,biocides,diagnostictools,reproductive
aids,andindrugandnutrientdeliveryinveterinarymedicine[13],andshowpotentialtoserveas
alternativestoconventionalantimicrobialagents[11]. Thus,itisnecessarytousenanotechnologyto
increasethesafetyofdomesticanimals,growth,production,andeliminatevariousdiseases,soasto
raisetheincomeoffarmers. Recently,theproductionoffoodsinthelivestockindustryusingdomestic
animalshasheavilyreliedontheuseofantibioticsasgrowthpromoters,leadingtogrowingconcern
overthespreadofmicrobialantibioticresistance. Theantibioticresistanceinbacterialeads,notonly
toaburdenonpublichealth,butalsoextendstotheriskoftherapyfailure,alongwithsubsequent
economicimpacts. Furthermore,themostsevereconsequenceofantibioticresistanceisthethreatof
importantendemicdiseasesinanimalskeptforfoodproduction[14]. Therefore,thedevelopmentof
innovativeandcost-effectivetherapeuticstrategiesisingreatdemandforthetreatmentofveterinary
animals. Inthisregard,nanoparticlesappeartobesuitableandalternativeantimicrobialagentsto
overcomethealarmingrateofthespreadofantibioticsresistance,towardimproveddetectionand
killingofpathogenicbacteria.Recently,severalstudieshavedemonstratedplantandmicrobialextracts,
essentialoils,puresecondarymetabolites,andnewlysynthesizedmoleculesaspotentialantimicrobial
agents[15–17].
Nanoparticle-basedtherapyisapromisingapproachtoimprovethebalancebetweentheefficacy
andtoxicityofsystemictherapeuticintervention. Amongthevariousmetalnanoparticlesavailable,
silvernanoparticles(AgNPs)haveattractedtremendousinterestinbiomedicalapplications,including
forantimicrobialtherapy,wounddressings,diagnosisandtreatment,andcontraceptivedevices[18].
Moreover,AgNPshavebeenusedassensors,imaging,drugdelivery,andfortissueengineeringin
veterinarymedicineandanimalproduction[19]. Thus,AgNPsrepresentaverypromisingtherapeutic
agentwithuniquepotentialagainstvariousmicrobialpathogens,withaparticularlyhighcapacityto
effectivelyactonantibiotic-resistantbacteria[11,20].Todate,AgNPshavebeenwidelyusedaseffective
antimicrobial agents against various bacteria, fungi, and viruses [21,22]. AgNPs can potentially
inhibitmultipledrug-resistantstrainsofStaphylococcusaureusandPseudomonasaeruginosathatcause
mastitis[20],andhaveproventobeeffectiveagainstvariousantibiotic-resistantbacteria[12,21,22].The
mechanismsofinhibitoryactionofAgNPsareattributedtotheirhighreactivitywithbacterialproteins,
sugars,andDNA,resultinginstructuralalterationstothecellwallandthemembrane,ultimately
leadingtoinhibitionandcelldeath[23].
Therefore,developingatherapeuticstrategybasedonAgNPstoenhancetheantibacterialeffect
representsanovelandpromisingapproach[24],particularlyintheeraofmultidrugresistance. Hence,
in the present study, we synthesized and characterized AgNPs using the biomolecule apigenin as
a reducing and stabilizing agent. Moreover, we isolated and characterized predominant isolates
fromclinicalendometritissamples,andevaluatedtheeffectofourdevelopedbiomolecule-assisted
AgNPs against multiple drug-resistant Gram-negative and Gram-positive bacteria, including
PrevotellamelaninogenicaandArcanobacteriumpyogenes. Finally,weevaluatedthemechanismoftoxicity
ofAgNPsinP.melaninogenicaandA.pyogenes.
2. ResultsandDiscussion
2.1. SynthesisandCharacterizationofAgNPsUsingApigenin
ApigeninwasreactedwithAgNO atpH8.0and40◦Cfor6h,andayellowbrownproductwas
3
observed,indicatingthatapigenincouldeffectivelyreduceAgNO intoAgNPs(Figure1).
3
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Figure1.Schematicdiagramofasimpleandenvironmentallyfriendlyapproachforthesynthesisof
silvernanoparticles(AgNPs)bythereductionofsilvernitratetoelementalsilverusingapigeninasa
reducingagent.
Theultraviolet–visiblespectrawereusedtodeterminethestructureoftheAgNPsbasedontheir
freesurfaceelectronplasmonoscillations. Theshiftingwavelength,likeblueandred,reflectthesize
and shape of the AgNPs [25,26]. The absorption of AgNPs strongly depends on the particle size,
dielectricmedium,andchemicalsurroundings. Smallsphericalnanoparticles(<20nm)exhibitasingle
surfaceplasmonband[25,26]. Thesynthesizedparticlesexhibitedmaximumabsorptionat407nm,
whichrepresentsthecharacteristicpeakforAgNPs(Figure2A).Inlinewiththesefindings,several
studieshavedemonstratedthatflavonoidsandphenoliccompounds,suchasquercetinandcaffeic
acid,canreduceAg+quickly,andcanacteffectivelyasbothreducingandstabilizingagents[23,27,28].
TheX-raydiffractionpatternoftheAgNPssynthesizedbyapigeninisshowninFigure2B.Several
strongBraggreflectionswereobserved,correspondingtothe(111),(200),and(220)reflectionsofface
centredcubic(fcc)silver[29]. Thehigh-intensitypeakofAg(111)wasobservedinthesample,which
indicatedthecrystallinenatureoftheparticles. ThediffractionpeaksofthesynthesizedAgNPspeaks
wereverysharp,andclearlysuggestedthattheAgNPssynthesizedusingapigeninarecrystallinein
nature[30]. Thesynthesizedparticleswerecrystalline,andthesizewasdeterminedtobe10nmusing
theDebye–Scherrerformula. AccordingtotheDebye–Scherrerformula,thepeakposition(111)reflects
thatthedimensionsoftheparticlesare10nm[31].
Fourier-transforminfrared(FTIR)measurementwascarriedouttoconfirmtheinvolvementof
variousfunctionalgroupsforreductionofAg+inapigenin,resultinginthecapping/stabilizationof
AgNPs. TheFTIRspectrumshowedabsorptionbandsat3422,2922,1742,and1042cm−1,indicating
the presence of a capping agent within the nanoparticles (Figure 2C). The band at 3422 cm−1 in
the spectrum corresponds to the O–H stretching vibration indicating the presence of alcohol and
phenol[28]. Bandsatthe2922cm−1regionwereobserved,arisingfromC–Hstretchingofaromatic
compound. Thebandat1743cm−1 wasassignedtoC–Cstretching. Severalstudieshavereported
that functional groups such as alcohol, phenol, and amines play a role in the stability/capping of
AgNPs [32]. The bands at 1042 cm−1 were assigned to N–H and C–N stretching vibrations of the
proteins,respectively[33]. Collectively,theFTIRdataconfirmedthatvariousfunctionalgroupsfound
inapigeninfacilitatethecappingandstabilizationofAgNPs.
Next,dynamiclightscattering(DLS)wasperformedtodeterminethesizedistributionofcolloidal
AgNPsintherangeof1–100nm. TheDLSmethodiswidelyusedinstudiesdealingwiththesynthesis,
functionalization,andbiomedicaluseofnanoparticlesthatexhibitplasmonresonance,primarilywith
regardtogoldandsilvernanoparticles[34].Thesynthesizedparticlesshowedanaveragesizeof20nm,
whichisthehydrodynamicsizeofanentirecomplex,ratherthanthegeometricalsizeofaparticle
itself(Figure2D).Ingeneral,thedispersionoftheDLSparticlenumbersizedistributionsnoticeably
exceedthesizedispersionobtainedfromtransmissionelectronmicroscopy(TEM)images[34]. We
further confirmed the particle size by TEM, which is a more reliable method for determining the
size and shape of particles. The TEM images revealed that most of the particles are significantly
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sphericalinshapewithasizeof10nm(Figure2E).ThehistogramofTEMimagesdeterminedfrom
thecalculationofseveralparticlesdemonstratedthattheTEMdataontheparticlenumberandsize
wereapparentlyaccurate,exhibitingthepresenceofanoticeablenumberofparticleswithdiameters
of10nm(Figure2F).Thus,thedataderivedfromDLSandTEMrevealedthatthemostpredominant
sizeoftheparticleswas10nm.
Figure 2. Synthesis and characterization of silver nanoparticles (AgNPs) using apigenin.
(A) Absorption spectrum of AgNPs synthesized using apigenin. (B) X-ray diffraction spectra of
AgNPs. (C)Fourier-transforminfraredspectraofAgNPs. (D)SizedistributionofAgNPsbasedon
dynamiclightscattering.(E)Transmissionelectronmicroscopy(TEM)imagesofAgNPs.(F)Histogram
displayingpredominantsizeofAgNPs.
2.2. Isolation,Identification,andCharacterizationofBacteriafromEndometritisSamples
Bacterialisolatesobtainedfromendometritissampleswerecultured,identified,andcharacterized
asdescribedintheMaterialandMethods. Among40swabs,20werefoundtobebacteriologically
positive by characterization of bacteria both phenotypically and biochemically [35,36]. The most
frequentlyisolatedbacteriumwasPrevotellamelaninogenica(30%),followedbyArcanobacteriumpyogenes
(25%),Escherichiacoli(20%),Streptococcusspp. (15%),Staphylococcusspp. (10%),Campylobacterfetus
(8%),Klebsiellaspp. (5%),P.aeruginosa(3%),andClostridiumspp. (1%). Similarly,Udhayaveletal.[37]
reportedthatoutof30samplesevaluated,25exhibiteddifferentstrainsofbacteria,includingE.coli
(36.66%),Klebsiellaspp. (30%),Proteusspp. (13.33%),P.aeruginosa(6.66%),andClostridiumspp. (3.33%).
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Sharmaetal.[36]reportedthatthemostfrequentlyidentifiedbacteriaisolatesfromuterinedischarge
samplesincludedE.coli(32.26%),Bacilluscereus(22.58%),S.aureus(16.13%),andmixedculturesof
B.cereusandS.aureus(9.68%),andE.coliandProteusvulgaris(3.23%). Altogether,ourdataagreewith
previousfindingsandindicatethatP.melaninogenicaandA.pyogenesrepresentthemostdominant
bacterialisolatesfoundinendometritisclinicalsamplesfromthedistrictofCoimbatore,TamilNadu.
2.3. IsolationofMDR
TheantibioticsusceptibilitytestwasperformedaccordingtoClinicalandLaboratoryStandards
Institute(CLSI)procedures. Weselectedmoreprofoundisolatesforfurtherantibioticsusceptibility
testing. Amongtheseveralisolatestested,MDRisolatesweredefinedasthoseshowingresistance
or intermediate susceptibility to more than three antimicrobials. The result of antimicrobial tests
showedthatalloftheisolatesofP.melaninogenicawereresistanttoampicillin(90.0%),cefalotin(79.0%),
sulfamethoxazole/trimethoprim (65.2%), ciprofloxacin (54.6%), oxolinic acid (45.4%), gentamicin
(43.8%), chloramphenicol(40.0%), cefotaxime(23.8%), ceftazidime(18.8%), amoxicillin/clavulanic
acid (10.0%), and aztreonam (5.0%). The A. pyogenes isolates exhibited resistance to all of the
antimicrobial agents tested, with particularly high levels of resistance found to chloramphenicol
(100%),amoxicillin(86.9%),ampicillin(76.1%),florfenicol(69.7%),penicillin(66.1%),oxytetracycline
(64.2%), and tetracycline (50%). Thus, the results from antibiotic susceptibility tests showed that
P. melaninogenica and A. pyogenes were resistant to at least three of the antimicrobial agents tested,
indicatingthattheseareMDRisolates. FurtherexperimentswerecarriedoutinP.melaninogenicaand
A.pyogenestoevaluatetheimpactofAgNPsonMDRbacteriainendometritis.
2.4. MinimumInhibitoryConcentration(MIC)andMinimumBactericidalConcentration(MBC)ofAgNPs
The MIC is the lowest concentration of AgNPs that will inhibit the visible growth of a
microorganismafterovernightincubation. TheMICwasdeterminedinbrainheartinfusion(BHI)
brothusingserialtwo-folddilutionsofAgNPsinconcentrationsrangingfrom0.1µg/mLto1.0µg/mL,
withanadjustedbacterialconcentrationof1×108 colonyformingunits(cfu)/mL(0.5McFarland’s
standard). MediumwithoutAgNPswasusedasacontrol. Theresultsfromthecellviabilityassay
suggestedthatAgNPsinhibitbacteriainadose-dependentmanner,andtheMICvaluesofAgNPs
against P. melaninogenica and A. pyogenes were found to be 0.8 and 1.0 µg/mL, respectively. The
MBC is the lowest concentration of AgNPs required to kill a particular bacterial strain. The MBC
values of AgNPs against P. melaninogenica and A. pyogenes were found to be 1.0 and 1.5 µg/mL,
respectively. AstheconcentrationofAgNPsincreasedtotheleveloftheMICoftherespectivestrains,
no growth was observed. The bactericidal effect of the AgNPs was dependent on several factors,
suchastheconcentrationofAgNPs,size,shape,physicochemicalproperties,andtheinitialbacterial
concentration. In general, AgNPs showed better antimicrobial activity against the Gram-negative
bacteriumP.melaninogenicawhencomparedtothatagainsttheGram-positivebacteriumA.pyogenes.
Our findings are consistent with previous reports suggesting that Gram-positive bacteria are less
susceptibletotheantimicrobialactivityofsilver[24,38,39].
2.5. Dose-andTime-DependentEffectofAgNPsonCellViabilityofP.melaninogenicaandA.pyogenes
To further promote the use of AgNPs in nanomedicine to overcome MDR in Gram-positive
andGram-negativebacteria,thedose-dependenteffectofAgNPswasassessedinP.melaninogenica
andA.pyogenestodeterminetheirrelativesusceptibilitiestoAgNPs,andtheextentofbactericidal
activity. Figure3Ashowsthepotentialtoxiceffectofapigenin-assistedAgNPsonP.melaninogenica
andA.pyogenes. Thebacterialstrainsweretreatedwithvariousconcentrations(0.2–1µg/mL)ofthe
10nmAgNPs. Theresultsshowedadosedependenteffectoncellviabilitycomparedtothenegative
control. Furthermore, cell viability decreased with increasing AgNPs concentrations. No visible
growth was observed at their respective MIC values (0.8 and 1.0 µg/mL) in P. melaninogenica and
A.pyogenes. InthecaseofP.melaninogenica,theintroductionof0.8µg/mLofAgNPsreducedbacterial
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viabilitybyapproximately95%,ascomparedtothatofthecontrolsample. Furthermore,increasingthe
concentrationofAgNPsto1µg/mLinhibitedbacterialgrowthdramaticallywithnovisiblegrowth
observed,whereasintroductionofasimilarconcentrationofAgNPs(i.e.,0.75µg/mL)reducedcell
viabilitybyapproximately75%ascomparedtothecontrolsample.However,thehigherconcentrations
of0.75and1.0µg/mLrapidlyinhibitedthegrowthofbacteria(Figure3A,B).
Figure3. AntibacterialactivityofAgNPsonP.melaninogenicaandA.pyogenes. (A)P.melaninogenica
andA.pyogeneswereincubatedwithvariousconcentrationsofAgNPs. Bacterialcellsurvivalwas
determined at 24 h based on a CFU count assay. (B) P. melaninogenica and A. pyogenes cells were
incubatedwith0.8and1.0µg/mLofAgNPs,respectively,for24h.
WepreviouslyreportedthattheantibacterialactivityofAgNPswithanaveragesizeof10nm
producedfromthecellularextractofBacilluscereusrequireda10-foldhigherconcentrationtoexhibit
asimilarantibacterialeffectagainstEscherichiafergusoniiandStreptococcusmutans, whichisdueto
thetypeofreducingagentsandtypeofbacteria[40]. Forinstance, AgNPscoatedwithlipoicacid
andpolyethyleneglycolexhibitedlowercytotoxicityascomparedwithAgNPscoatedwithtannicin
agingivalfibroblastmodel[41]. Strydometal.[42]suggestthatmodificationofsilversulfadiazine
usingdendrimersdisplayedpotentialantibacterialactivity. TheantimicrobialactivityofAgNPsalso
dependsonthesurfacearea,whicheffectivelyinteractswithacertainmicroorganism. Severalstudies
havesubstantiatedthatauniquefeatureoflargesurfaceareaofnanoparticleshavethesignificant
possibilityinteractwithmicrobes[21,43].
2.6. Dose-andTime-DependentEffectofAgNPsontheBiofilmActivityofP.melaninogenicaandA.pyogenes
Toexaminetheanti-biofilmactivityofAgNPsonP.melaninogenicaandA.pyogenes,thebacteria
weregrownintissuecultureplatesinthepresenceandabsenceofAgNPsfor24h. Bothbacterial
strainsweregrownfor24hinmicrotiterplatewellsandthentreatedwith0.1–1.0µg/mLAgNPs
(Figure 4A). AgNPs decreased the biofilm activity of P. melaninogenica and A. pyogenes by more
than 95% and 90%, respectively. Our findings are consistent with previous reports in various
Gram-negativeandGram-positivebacteria. Interestingly,AgNPsinhibitedbiofilmformationfaster
within 20 h in P. melaninogenica than in A. pyogenes, which is likely due to the structural nature of
thecellwallandmembrane(Figure4B).Bacteriabiofilmsareresistanttoantibiotics,disinfectants,
and components of the innate and adaptive inflammatory responses [26,44]. AgNPs potentially
inhibit cell viability and biofilm formation against P. aeruginosa and Staphylococcus epidermidis by
inhibitingproductionofexopolysaccharides,whichareessentialforbiofilmformation[25,45]. Plant
extract-mediatedsynthesisofAgNPsefficientlyinhibitedbiofilmformationinHelicobacterpyloriand
Helicobacter felis [38]. Martinez-Gutierrez et al. [46] demonstrated the quorum-quenching activity
of AgNPs against various Gram-negative and Gram-positive bacteria. Taken together, our results
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suggestthattheapigenin-mediatedsynthesisofAgNPscouldbeapotentialandviablealternative
anti-biofilmagent.
Figure4.Anti-biofilmactivityofAgNPsonP.melaninogenicaandA.pyogenes.(A)P.melaninogenicaand
A.pyogeneswereincubatedwithvariousconcentrationsofAgNPs.Anti-biofilmactivitywasmeasured
using96-wellflat-bottompolystyrenetissuecultureplates.(B)P.melaninogenicaandA.pyogenescells
wereincubatedwith0.8and1.0µg/mLofAgNPsrespectivelyfor24h.
2.7. AgNPsInduceMetabolicToxicityinP.melaninogenicaandA.pyogenes
Perturbationsofmetabolicactivityareapossiblestrategytoimpacttheefficacyofantimicrobial
therapy. Lactateisaveryimportantendproductofcarbohydratessynthesisinbacteria. Toevaluate
theeffectofAgNPsonoxidativestress-inducedmetabolicchanges,alactatedehydrogenase(LDH)
assaywasperformedincellsexposedtoAgNPsfor12h[47]. AsshowninFigure5A,thelevelofLDH
inP.melaninogenicaandA.pyogeneswasfour-foldhigherthanthatofthecontrolgroup. Althoughboth
bacteriaexhibitedsimilarlevelsofLDH,thatoftheGram-positivebacteriumA.pyogeneswasslightly
lowerthanthatoftheGram-negativebacteriumP.melaninogenica,whichisduetothearchitectureof
thecellwallandmembrane. Ourresultsclearlydemonstratedthattheactivitiesofrespiratorychain
dehydrogenases(RCD)inbothP.melaninogenicaandA.pyogeneswereinhibitedbyAgNPs,whichis
inlinewithpreviousstudiesdemonstratingthemechanismofantimicrobialaction[23,48–51]. One
possiblemechanismunderlyingthismetabolicdisturbanceistheentryofAgNPsintothecellstoRCD
andalterdissolvedoxygenlevelsinculture[45]. AnotherpotentialmechanismisthattheAg+ofthe
AgNPsinteractwiththethiol(–SH)groupofcysteine[51].
Usinginsilicogenome-scalemetabolicmodels,Brynildsenetal.[52]clearlydemonstratedthatan
increaseintheintracellularproductionofendogenousreactiveoxygenspecies(ROS)couldagitatethe
productionandusageofATP.ATPisanenergy-richmoleculethatgovernsvariouscellularfunctions
suchassurvival,growth,andreplication,andactsasamajorsignalingmolecule[53]. Inlinewith
that prediction, we sought to determine the level of ATP in AgNPs-treated P. melaninogenica and
A.pyogenes. ThelevelofATPinAgNPs-treatedsampleswassignificantlylowerbyuptofive-fold
compared to that of the control samples (Figure 5B), indicating that AgNP-induced cellular stress
significantlyaffectsATPsynthesisinP.melaninogenicaandA.pyogenes,whichisacriticalfactorfor
bacterialgrowthandreproduction[25]. AgNPsdirectlyaffectFOF1-ATPaseactivityandH+-coupled
transport[54]. FOF1-ATPaseplaysacrucialroleincellmetabolicprocesses,includingbacterialgrowth,
metabolicregulation,andcellsurvival. Therefore,thedatafromthepresentstudyandtotalbodyof
previousworksuggestthatmetabolicactivitycontributingtoATPproductionisanintegralpartofthe
bactericidaltoxicityofAgNPs.
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Figure5.MetaboliccytotoxicityofAgNPsonP.melaninogenicaandA.pyogenes.P.melaninogenicaand
A.pyogenescellswereincubatedwith0.8and1.0µg/mLofAgNPsrespectivelyfor12h, andthe
(A)LDHactivity,(B)ATPlevels,(C)proteinlevels,and(D)sugarlevelsweredetermined.
To validate the effect of AgNPs on the weakening of metabolic activity, we further explored
thelevelsofproteinsandsugars. WepreviouslydemonstratedthatAgNPsarepotentialagentsto
increaseproteinleakagebyalteringthemembranepermeabilityinbacteria[40]. P.melaninogenicaand
A.pyogenesweretreatedwith0.8and1.0µg/mLofAgNPs,respectively,andtheamountofprotein
releasedinthesuspensionwasestimatedusingtheBradfordassay. TheresultsshowedthatAgNPs
remarkablyincreasedtheleakageofproteinscomparedtothecontrolgroup(Figure5C).However,the
leakagefromP.melaninogenicacellstreatedwithAgNPswassignificantlyhigher(60µg/mg)thanthatof
A.pyogenes(40µg/mg),suggestingthattheantibacterialsensitivityofGram-negativebacteriaismuch
strongerthanthatofGram-positivebacteria. Similarly,Kimetal.[51]andGurunathanetal.[24]found
thattheleakageofproteinswassignificantlyhigherinGram-negativebacteriathaninGram-positive
bacteria with 60 and 50 µg of reducing sugars leaking from P. melaninogenica and A. pyogenes
treatedwithAgNPs,respectively(Figure5D).ApreviousstudyshowedthatafterE.colicellswere
exposedtoAgNPs(10µg/mL)for2h,upto102.5µgperbacterialdryweightof1mgofreducing
sugarsleakedoutofthecells[49]. Thisdifferentialleakageamountcouldbeduetothestructural
features of the cell wall of A. pyogenes, which is essential for protecting the bacteria various toxic
agents[23,24,51]. TheimpairmentofthefunctionofLDHcouldleadtoincreasedleakageofproteins
and other macromolecules. Altogether, all of the available evidence from various bacteria clearly
indicate that AgNPs could alter membrane permeability and eventually damage the structure of
thebacteriacellmembranebyosmoticimbalance,resultingintheleakageofmacromoleculessuch
as proteins and reducing sugars, leading to the death of bacteria. This mechanism highlights the
significantpotentialoftheantibacterialactivityofAgNPs.
2.8. AgNPsInduceCellularToxicityandOxidativeStressinP.melaninogenicaandA.pyogenes
TounderstandtheeffectsofAgNPsoncellviabilityandmetabolictoxicity,wefurtherexamined
how the influence of AgNPs onbacterial metabolism could offerinsight into theirmechanisms of
action,leadingtoenhancedtherapeuticapproachesforbothhumansandanimals. Majorclassesof
bactericidalantibioticsinducecelldeathinbacteriabystimulatingtheproductionofhighlydeleterious
hydroxylradicals[55]. AsimilarmechanismhasbeendemonstratedforAgNP-inducedcelldeathin
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avarietyofbacteria,includingthemostrepresentativeGram-negativeandGram-positivebacteria,
such as P. aeruginosa, Shigella flexneri, S. aureus, and Streptococcus pneumonia [24]. However, to our
knowledge, nostudyhasdemonstratedthemechanismofAgNPsontheoxidativestress-induced
celldeathinP.melaninogenicaandA.pyogenes. ThebacteriaweretreatedwiththerespectiveMICof
theAgNP,andROSgenerationwasmeasuredusingthe2(cid:48),7(cid:48)-dichlorofluorescindiacetate(DCFDA)
assay. TheresultsindicatedthatAgNPsinducedtwo-foldhigherlevelsofROSinP.melaninogenica
andA.pyogenescomparedtothecontrol(Figure6A).AnincreasedlevelofROSleadstoanimbalance
betweenpro-oxidantsandantioxidants,whichcausesfailureinnormalphysiologicalredox-regulated
functions[55]. Indeed,ROScanbeinducedbyvariousexternalsources,suchaschemicals,antibiotics,
nanoparticles,andcoldandheatstress,consequentlyleadingtolossofcellviability[33,40,55–57].
Figure6.EffectofAgNPsoncellulartoxicityinP.melaninogenicaandA.pyogenes.(A)P.melaninogenica
and A. pyogenes cells were treated with 0.8 and 1.0 µg/mL of AgNPs respectively for 12 h. ROS
generationwasmeasuredusingDCFDA.(B)MDAlevelsweremeasuredusingaTBARSassay.(C)The
relativeproteincarbonylcontentwasevaluatedcomparedtothetotalproteincontent.(D)Thequantity
ofNOwasquantifiedspectrophotometricallyusingtheGriessreagent.
Next, we examined the level of malondialdehyde (MDA), which is a well-known marker in
eukaryoticcellsforoxidativestress,asitisgeneratedfromlipidsbystimulationofoxidativestress.
ToascertaintheMDAlevelsinAgNP-treatedbacteria,weusedthiobarbituricacid. Treatmentwith
AgNPsledtoincreasedlevelsofMDAbyseveralfoldinP.melaninogenica,comparedtothecontrol
group(Figure6B);similarincreaseswerealsoobservedinA.pyogenes. Thesefindingssuggestthat
lipidoxidationinducedMDAproductioninbacteria. Belenkyetal.[58]foundthatantibiotic-treated
E.colicellsexhibitedcytotoxicchangesthatwereindicativeofoxidativestress,includinghigherlevels
of protein carbonylation. The carbonylation of proteins could lead to protein dysfunction [55–57].
Therefore,wehypothesizedthatAgNPscouldtargetthewell-knownoxidativestressbiomarkerof
carbonylation. Tomeasurethecarbonylcontent,P.melaninogenicaandA.pyogenesweretreatedwith
AgNPs for 12 h, which led to significant increases in protein carbonylation, up to 12 times above
thatofthecontrol(Figure6C).Thesefindingsareconsistentwiththeeffectsofbacteriatreatedwith
Ampicillin(Amp),kanamycin(Kan),orNor[58]. Nitricoxide(NO)producedbybacterialnitricoxide
synthase(NOS)actsasacytoprotectiveagentagainstoxidativestressinS.aureus,Bacillusanthracis,and
Bacillussubtilis[58]. ToexplorewhetherAgNPsinducetheproductionofNOorinhibit,weexamined
Int.J.Mol.Sci.2018,19,1210 10of20
theeffectofNOproductioninAgNP-treatedP.melaninogenicaandA.pyogenes. AgNPsinducedthe
productionofNOinbothP.melaninogenicaandA.pyogenes(Figure6D).Interestingly,NOproduction
wassignificantlyhigherintheGram-negativebacteriumthanintheGram-positivebacterium,which
indicatesthatP.melaninogenicamaybemoreimmunetothestresscreatedbyAgNPs. Previousstudies
demonstrated that the oxidative stress generated by AgNPs was associated with reduction in the
levelsofreactivenitrogenintermediatesinbacteriatreatedwithdifferentantibiotics[59]. Collectively,
thepresentstudysuggeststhatAgNPsinteractwithbacterialcellsviathecellwallandmembrane,
resulting in the production of free radicals to, in turn, induce oxidative stress and cause various
dysfunctionstomacromolecules,includinglipids,proteins,andnucleicacids[22,60–63].
2.9. EffectofAgNPsontheExpressionofAntioxidativeMarkersinP.melaninogenicaandA.pyogenes
The antioxidative stress response counteracts the effect of pro-oxidants to maintain normal
physiological redox-regulated functions. Masip et al. [64] demonstrated that a depressed ratio of
reducedglutathione(GSH)tooxidizedglutathione(GSSG)isconsideredanindicatorofoxidative
stress. Thus,thelevelsofGSHandGSSGweredeterminedinP.melaninogenicaandA.pyogenestreated
withAgNPsfor12h,demonstratingdecreasedlevelsofGSHcoupledwithhighlysignificantdecreases
inGSSG(Figure7A,B).ThisdecreasedlevelofGSHintheAgNP-treatedcellssuggestsaninabilityto
protectthecellsfromoxidativestress,sothatthecellsweresubjectedtocelldeathduetooverwhelming
oxidativestress.Banerjeeetal.[65]observedincreasedlevelsofoxidativestressanddecreasedlevelsof
antioxidantsinE.colicellstreatedwithaniodinatedchitosan–silvernanoparticlecomposite. Similarly,
E.coliandP.aeruginosatreatedwithAgNPsexhibitedasimilartrend[20,63]. Together,thesechanges
inmetabolitelevelssuggestthatdecreasedGSHisunabletocompensatefortheongoingturnoverand
consumptionbypro-oxidantactivities. Collectively,thesedatasuggestthatthecomplexmetabolic
changesofAgNPsareinducedbyoxidativestress.
Figure7.EffectofAgNPsonantioxidants.P.melaninogenicaandA.pyogenescellsweretreatedwith0.8
and1.0µg/mLofAgNPsrespectivelyfor12h,andthe(A)GSHlevels,(B)GSSGlevels,(C)superoxide
dismutase(SOD)activity,and(D)catalase(CAT)activityweremeasuredasdescribedintheMaterials
andMethods.
Silverionsareresponsiblefortheformationoffreeradicals.Therefore,wenextexaminedenzymes
withantioxidanteffectssuchassuperoxidedismutase(SOD)andcatalaseinP.melaninogenicaand
A.pyogenestreatedwith0.8and1.0µg/mLofAgNPsfor12h.AgNPsdecreasedthelevelofSODbyup