Investigation of the Efficacy of Synthesized Silver and Zinc … · Investigation of the ... antibacterialactivity of Ag-NPs and ZnO-NPs against bacterial pathogens, no available

2017Vol.8 No.5:67

Research Article

1© Under License of Creative Commons Attribution 3.0 License | This article is available from: www.acmicrob.com

ARCHIVES OF CLINICAL MICROBIOLOGYISSN 1989-8436

iMedPub Journalswww.imedpub.com

DOI: 10.4172/1989-8436.100067

Maha Abd El Fattah K1,2*, Nanis Gamal A1, Fatma Ibrahim S3, Gamal Mohamed ELM3 and Perihan Saleh A2

1 Department of Botany, Tanta University, Tanta, Egypt

2 Department of Biology, Faculty of Science, Taif University, Taif, KSA

3 Microbiology Department, Faculty of Pharmacy,Tanta University, Tanta, Egypt

4 PharmaceuticaltechnologyDepartment,Faculty of Pharmacy, Tanta University, Tanta, Egypt

*Corresponding author: MahaAbdElFattahKhalil

[email protected]

Department of Biology, Taif University, Taif, KSA.

Tel: +966560869438Fax: 027274299

Citation: FattahKMAE,GamalAN,IbrahimSF,MohamedELMG,SalehAP(2017)InvestigationoftheEfficacyofSynthesizedSilverandZincOxideNanoparticlesagainstMulti-DrugResistantGramNegativeBacterialClinicalIsolates.ArchClinMicrobiol.Vol.8No.5:67

Investigation of the Efficacy of Synthesized Silver and Zinc Oxide Nanoparticles against

Multi-Drug Resistant Gram Negative Bacterial Clinical Isolates

AbstractBackground/aim: The increasingnumberofmultidrug-resistant (MDR)bacteriaand the need to synthesize new antimicrobials, nanoparticles have attractedinterestinthescientificcommunity.ThisstudyaimedtoevaluatethesynergisticeffectofnanoparticleswithsomeantibioticsagainstMDRpathogenicbacteria.

Material and methods: Seventeen bacterial isolates including; Escherichia coli (n=5) and Klebsiella pneumoniae (n=12) were collected from different clinicalsamplesofpatientssufferingfromburnwoundandfistulaewoundsinfections.Thesusceptibilityofthecollectedisolatestodifferentantibioticswasinvestigated.SixMDRisolateswereselectedasrepresentativestodifferentpatternswereusedforthedeterminationoftheantibacterialactivitiesoftwometalsnanoparticles;silver(Ag-NPs)andzincoxide(ZnO-NPs).Theantibacterialactivitiesofthesynthesizednanoparticles alone and their combination with the selected antibiotics weredeterminedagainstthetested isolatesusingdisc-diffusionmethodaccordingtoEUCASTClinicalBreakpoint.

Results:WelldispersedsphericalAg-NPswithaverageparticlesize12.65±0.55nmandZnO-NPsof7.6±0.5nmwere chemically synthesizedby the chemicalreductionmethodandchemicalprecipitationmethod,respectively.Theobtainedresults suggested that the Ag-NPs alone had higher antibacterial activity thanZnO-NPs.Furthermore,Ag-NPsshowanobservablesynergisticeffectwithsomeantibiotics;morethanZnO-NPs.

Conclusions: It was concluded that Ag-NPs have potential as a combinationtherapeuticagentforthetreatmentofinfectiousdiseasesbybacteria.

Keywords: MDR bacteria; Ag-NPs; ZnO-NPs; Antibiotics-nanoparticlescombination;Antibacterialactivity

Received: September 18, 2017; Accepted: September 29, 2017; Published: October 12, 2017

IntroductionMicrobiologists and pharmacists are in continuous challengeto deal with, control and treat diseases caused by microbeswe face daily. Recently the global public health is in a seriousthreatwiththeincreasinglyintroducedantimicrobialresistanceof the infecting pathogens to the ordinary known treatments.The antimicrobial resistance is defined as the resistance of amicroorganism to an antimicrobial drug that was originallyeffectiveforthetreatmentof infectionscausedby it.Resistant

microorganisms such as bacteria, fungi, viruses and parasites are able to withstand the attack by antimicrobials such as;antibacterial (antibiotics), antifungal, antiviral and antimalarialdrugs, respectively [1]. As a result, those standard treatmentsbecomeineffectiveandinfectionspersistwithprolongedillness,higherhealthcareexpendituresandgreaterchancefordeathwiththeriskofspreadingtotheothers[2].Itisimportanttomentionthat the antimicrobial resistance's development is presentin either developing or advanced countries with impressiveincreaseinthenumbersofvictimswhodiedbyinfectionscaused

2017Vol.8 No.5:67

2 This article is available from: www.acmicrob.com


infectionandfistulaewounds.The clinical samples used for this studywerecollectedfrompatientsattendingdifferenthospitalsincluding; Burn unit of Tanta Emergency Hospital, Mubarak Hospital and El-Salam Hospital in Tanta, Egypt. Each specimen wasculturedonbloodagarandnutrientagarplates.Thegrowingcolonies on these media were subcultured on MacConkeyagar medium and Eosin methylene blue agar (EMB-agar). TherecoveredisolatesweresubjectedtodifferentmorphologicalandbiochemicaltestsforidentificationtothespecieslevelasdescribedbyBergey’sManualofDeterminativeBacteriology9thedn.[8].

The isolates were identified as Escherichia coli (n=5) andKlebsiella pneumoniae (n=12). Identification of tested isolateswas confirmedwith an API 20E (bioMérieux) identification kit.Stockcultureswerestoredin0.05MK-Na-phosphatebuffer,pH7.0,containing15%glycerolat-20°C.

AntibioticsFor studying the combination effect of metal-nanoparticleswithantibiotics;theantibioticsusedinourstudywereselectedto cover almost all the different antibiotic classes suitable forEnterobacteriaceae isolates. The antibiotics used with theirantimicrobial subclasses along with the discs' potencies arepresented in Table 1.

Chemical synthesis of metal nanoparticlesSynthesis of Ag-NPs by the chemical reduction method: The Ag-NPsused in this studyweresynthesizedusing thechemicalreduction method [9]. This method involves the reduction ofsilvernitrateaqueoussolution(AgNO3)byusingtrisodiumcitrate(C6H5O7Na3)which acts as reducing agent and stabilizing agent[10].AgNO3andC6H5O7Na3(SigmaAldrich,UK)ofanalyticalgradepuritywereusedasstartingmaterials.Inthatmethod,50mlof1× 10-3 MAgNO3washeatedtoboiling.Later,5mlof1%C6H5O7Na3 wasaddeddropbydrop to this solutionwith vigorousmixing.The mixture was heated until color change was evident (paleyellow)indicatingtheformationofAg-NPs.Thesuspensionwasremoved from theheating element and stirreduntil cooled toroom temperature.

by resistant microorganisms every year. Melake et al. [3]studiedtheprevalenceofMDRbacterialisolatescausingburninfectionsfrom105patientsadmittedtheBurnUnitofMenoufiaUniversityHospital. They reported that about 36% of patients had burnwound infections and about 63% of the bacterial growth wasMDR isolates.

Although, resistance of bacteria to antibiotics could occurnormally as a natural phenomenon, certain human actionsaccelerate its emergenceand spreadwhere; the inappropriateuse of antibiotics either by overuse ormisuse can lead to theevolution of new MDR bacteria. If these trends persist andresistance continues to rise, some reports estimated that by2050 there will be ten million antimicrobial resistance relat-eddeathsworldwide,costingtheworldupto100trillion$(2).Unfortunately,thediscoveringofnewantibioticsisconsideredarealchallengetotheresearchersandpharmaceuticalcompaniesdue to the difficulty of identifying novel bacterial targets thatcould be used for the innovation of new classes of safe andeffectiveantimicrobialagentsbya ratecovering the incredibleincreaseinbacterialresistanceamongtheworld [4].

Nowadays,thereisagreatinterestinintegratingnanotechnologywithbiologyinordertodevelopnewantimicrobialdrugsbasedon nanotechnology with higher affectivity. It was reportedthat some noblemetals like gold and silver exhibit interestingantibacterial activity [5-7]. However, only few studies havecompared the efficacy of noble metals nanoparticles such assilver nanoparticles (Ag-NPs) with that of other metals oxideslikeZinc-oxide(ZnO-NPs).Alsotheeffectofthosenanoparticleson enhancing the activity of different antibiotics belonging todifferent antimicrobial classes is still under investigation. It isimportant to note that although there are some reports on the antibacterial activity of Ag-NPs and ZnO-NPs against bacterialpathogens, no available study has been done to evaluate the synergisticpotentialofthosenanoparticleswithantibioticswithrespecttotheknownCLSIstandards.Thepresentstudyaimstotested whether the combination of synthesized nanoparticleswiththeselectedantibioticscanposedistinguishingantibacterialefficacy against tested clinical isolates, when compared toantibiotic alone,whichmight have implications on the controland treatment of infections. The susceptibility of the selectedisolates to antibiotic-nano combination according to EUCASTClinical Breakpoint was performed. Consequently detectinghowever the isolates became more susceptible to antibioticafter nano-combination or the combination resulted in onlyslight increase in the inhibition zone whiles the organism stillresistant. Furthermore, the nanoparticles were characterizedusing different chemical techniques including; UV-visibleSpectrophotometer, Transmission Electron Microscopy (TEM)and Energy dispersiveX-rayanalysis(EDX).

Materials and MethodsIsolation and identification of clinical bacterial isolatesAtotaloftwentyfiveclinicalspecimenswererandomlycollectedinsterilescrewcappedcontainersfrompatientswithburnwound

Antimicrobial class Antimicrobial agent Disc contentPenicillins Ampicillin(Am) 10 µg

β-lactam/β-lactamaseinhibitorcombinations

Amoxycillin/Clavulanicacid(20/10µg)(AMC) 30 µg

Cephems Cefotaxime(CTX) 30 µgMonobactams Aztreonam(ATM) 30 µgCarbapenems Imipenem(IPM) 10 µg

Aminoglycosides Gentamycin(GEN) 10 µgQuinolones Levofloxacin(LEV) 5 µg

Foliatepathwayinhibitor Trimethoprim-sulfamethoxazole(SXT) 25 µg

Phenicols Chloramphenicol(C) 30 µgTetracyclines Tetracycline(TE) 30 µg

Table 1Antibioticsusceptibilitydisks(Bioanalyse)alongwiththeircodesand potencies.

2017Vol.8 No.5:67


3© Under License of Creative Commons Attribution 3.0 License

The mechanism of reaction could be expressed according toŠileikaitėetal.[11]asfollows:2Ag++C6H5O7Na3 + 2 H2O→4Ag0

+C6H5O7H 3+3Na+ + H+ + O2↑

Synthesis of ZnO-NPs by precipitation method: ZnO-NPsweresynthesizedbytheprecipitationmethoddescribedbySalahuddinet al. [12]intheChemistryDepartment,FacultyofScience,TantaUniversity. Zinc nitrate hexahydrate (Zn (NO3)2.6 H2O) (0.1 M,solution A) and sodium carbonate (Na2CO3) (0.12 M, solutionB)werepreparedthensolutionAwasaddedtosolutionBdropwiseundervigorousstirring.Thewhiteprecipitateformedwascollectedbyfiltrationandrinsedwithdistilledwaterthreetimes.Thesolidwasthenwashedwithethanolanddriedat100°Cfor6h.

Characterization of the chemically synthesized nanoparticlesVisual inspection: In case of Ag-NPs, the conversion of thecolorless solution of AgNO3 to a pale yellow color after thereductionwithsodiumcitrateclearlyindicatedtheformationofAg-NPs[9].While,incaseofZnO-NPs,theformationofawhiteprecipitate from the precipitation of zinc nitrate indicated theformationofZnO-NPs[12].

UV-Vis spectroscopy: The formation of the nanoparticlesand their optical properties were analyzed via UV-visibleSpectrophotometer according to the method of Mie [13] atwavelength 300-800 nm with Ag-NPs and 360-500 nm withZnO-NPsusingShimadzudualbeamspectrophotometer(ModelUV1650PC)operatedata resolutionof1nm in theChemistryDepartment, Faculty of Science, Tanta University, Egypt.

Transmission electron microscopy (TEM):Themorphologyandthe mean size of the synthesized Ag-NPs and ZnO-NPs weremeasuredby JEOL, JEM-100 SXelectronmicroscope (Japan) inFaculty of Medicine, Tanta University. The transmission electron microscopy was operated at 200 kv and the samples werepreparedbydroppingthenanoparticlesontocarboncoatedCugridunderlyingtissuepaper,leavingbehindafilm[10].

Energy dispersive x-ray analysis (EDX): In order to detectthe elemental composition of the synthesized nanoparticles,their nano-powderswere analyzedby EDX [14]. SampleswerecollectedinthepowderformandanalyzedbyEDX(modelFEI)intheChemicalWarLabs,ArmedForces,Cairo,Egypt.

Assessment of antibiotic susceptibility, resistance pattern and calculating the multiple antibiotics resistance (MAR) index of the tested bacteriaAlltheisolatesweretestedfortheirsusceptibilitytotheselectedantibioticsusingDiscDiffusionMethod[15].Bacterialinoculum(100µl)of1×108CFU/mlforeachisolatewasusedtoinoculateMueller-Hintonagar(MHA).Theplateswereallowedtosetandthe selected antibiotic discs were placed using sterile forcepsontotheagarplates.Thediametersoftheinhibitionzonesweredetermined and the results were interpreted as susceptible,intermediately resistant or resistant by referring to the PerformanceStandardsforAntimicrobialSusceptibilityTesting[16].

The resistance patterns of the isolateswere recorded and theMARindexwascalculatedforeachisolate[17,18] asfollow:

Numberofantibioticsisolatesareresistantto

MARindex=----------------------------------------

Totalnumberofantibioticstested

Determination of the antibacterial efficacy of the synthesized nanoparticles The antibacterial activity of synthesized Ag-NPs and ZnO-NPswasinvestigatedbywelldiffusionmethodusingsixisolates;(twoisolates of E. coli and four isolates of K. pneumoniae) selectedrandomlyas representatives to thedifferent resistantpatternsobtained.Thetestedinoculumswereadjustedtoconcentration1 × 108CFU/mlthen100µlofeachinoculumwasappliedontothesurfaceofMHAplatesfreeofnanoparticlesusingsterilecottonswab.Theplateswereallowedtodryandasterilewell-cutterofdiameter6.0mmwasusedtoboretwowells ineachplate.Subsequently,twovolumes(50μland100μl)oftheAg-NPsorZnO-NPs suspensions were introduced, respectively into thewells.Theplateswereallowedtostandfor1hforthediffusiontotakeplaceandthenincubatedat37°Cfor18h.Afterincubation,thediametersoftheinhibitoryzones(mm)weremeasured[19].Allthestepsweremadeintriplicates.

Determination of the efficacy of nanoparticles on enhancing the activity of different antibioticsOnly antibiotics to which the selected isolates were found tobe resistant were selected for investigation. The antibacterialactivitiesof these antibiotics alonewere comparedwith thosewiththeircombinationswithAg-NPsandZnO-NPs,respectivelyby disc diffusion method against the selected isolates [5,20].Bacterialinoculawerepreparedandtheplateswereinoculatedasmentionedbefore.Theplateswereallowedtosetandthenthe standard antibiotics’ discs with and without nanoparticleswereplacedontothesurfaceoftheinoculatedagarplatesusingsterileforceps.Fordeterminingthesynergisticeffectsbetweenantibioticsandnanoparticles,antibioticdiscs impregnatedwith50µlofAg-NPsorZnO-NPs,respectivelywereused.Plateswerelabeledcarefullyandincubatedfor18hat37°C.Afterincubation,the diameters of the inhibitory zones (mm) around the discsweremeasured.Allexperimentswereconductedintriplicatestoconfirmtheresults.

The fold increase in the diameter of inhibition zone of eachantibiotic after the combination with Ag-NPs or ZnO-NPs wascalculatedaccordingtotheequation;

Thefoldincrease=(b2-a2)/a2,

Where;(a)istheinhibitionzoneofantibioticaloneand(b)istheinhibitionzoneofantibioticplusnanoparticles[20].

Thenumberofisolateschangedfromresistanttoanantibiotictointermediatelyresistantorsensitivetothesameantibioticafterthecombinationwithnanoparticleswererecorded.

2017Vol.8 No.5:67



Results and DiscussionChemical synthesis and characterization of nanoparticlesInthisstudy,twodifferentmetalsnanoparticlesincludingsilvernanoparticles (Ag-NPs) and zinc oxide nanoparticles (ZnO-NPs)werechemicallysynthesizedandanalyzedbymultipletechniquesin order to insure the completion of the nano formation anddetectionoftheircharacteristics.

Visual inspectionThesynthesisofAg-NPswasdetectedbyvisualobservationfortheconversionofthecolorlesssolutionofAgNO3 toapaleyellowcolorby thereductionwithsodiumcitrate [9].WhileZnO-NPs,wasindicatedbytheformationofawhiteprecipitate(Figure 1)[20].

UV-Vis spectroscopy: The UV–Vis absorption spectrum of thesynthesizedAg-NPs is shown inFigure 2where,awelldefinedsurfaceplasmonbandcenteredataround420nm,characteristictoAg-NPswasobtained.ForZnO-NPs,ChiengandLoo [21]reportedthatthebroadabsorptionpeakobservedinspectrumat360-380nm isacharacteristicband for thepureZnOnanoparticles.Nootherpeakwasobserved in the spectrumwhich confirms thatthesynthesizedproductsareZnOonlyFigure 3.

Energy dispersive x-ray analysis (EDX): TheEDXprofilesobtainedinthepresentstudyconfirmedthepresenceofAg-NPsandZnO-NPswithstrongsignalenergypeaksforAgandZnatomsinthestandardrangeasshowninFigures 4 and 5.

Transmission electron microscopy (TEM): The morphology and theaveragesizeofthesynthesizednanoparticleswereanalyzedby Transmission Electron Microscopy(TEM).TheparticlesofAg-NPssynthesizedbythechemicalreductionmethodandZnO-NPssynthesized by precipitationmethod arewell separated singlespherical particles without aggregation. The presence of theparticleswithoutaggregationclearlyinsuredtheefficiencyofthetechniquesusedinthesynthesizingprocesses.Theaveragesizefortheparticleswascalculatedandfoundtobe12.65±0.55and7.6±0.5nmforAg-NPsandZnO-NPs,respectively(Figure 6).

Antibiotic resistance pattern and MAR index of the isolated bacteria.In the present study a total of seventeen isolate;E. coli (n=5)and K. pneumoniae (n=12) isolates were recovered from

clinical specimens.Thesusceptibilityof the tested isolateswasperformed against 10 different antibiotics using disc diffusionmethod.The incidenceof resistancetotheselectedantibioticswas presented in Table 2. It was found that all the isolateswere resistant to Ampicillin, Amoxicillin/ Clavulanic acid andCefotaxime. Moreover, 15 isolateswere found to be resistant

Figure 1 Ag-NPssynthesizedbythechemicalreductionofsilvernitrate salt (1) where; (A) silver nitrate solution, (B)colloidal Ag-NPs formed. ZnO-NPs synthesized by theprecipitationmethodassoliddryprecipitate(2).

Figure 2 Ultravioletvisible(UV-Vis)spectrumofthesynthesizedAg-NPs.

Figure 3 Ultravioletvisible(UV-Vis)spectrumsofthesynthesizedZnO-NPs.

Figure 4 EnergydispersiveX-ray(EDX)analysisofthesynthesizedAg-NPs.

2017Vol.8 No.5:67



toTrimethoprim/Sulfamethoxazole followedbyAztreonam (11isolates)andChloramphenicol(10isolates)thelowestresistanceamongtheisolateswasobtainedagainstLevofloxacinantibioticwhereonly4 isolateswere found tobe resistant.Noneof theisolates showed any resistance to Imipenem or Gentamycin.From the obtained data, itwas concluded that all the isolateswere highly MDR isolates. Abbas et al. [22] investigated thebacterialinfectionsinpatientswithburnwoundsinaburnunitin Hehia General Hospital in Egypt. Total of 160 isolateswererecovered; where; S. aureus was themost common pathogenbesides P. aeruginosa, K. pneumoniae and S. epidermidis E. coli, Enterobacter cloacae and Citrobacter freundiiwerealsopresent.MostoftheisolateswerehighlyMDRas100%oftheS. aureus

Figure 5 EnergydispersiveX-ray(EDX)analysisofthesynthesizedZnO-NPs.

Figure 6 TransmissionelectronmicrographsshowingsphericalwellseparatedanddispersedsynthesizedAg-NP(A)withaveragesize12.65±0.55andZnO-NPs (B)withaveragesize7.6±0.5nm.

i,k

A B

Antibiotic No. of resistant isolates (%)Ampicillin 17(100)

Amoxicillin/Clavulanic acid 17(100)Cefotaxime 17(100)Aztreonam 11(64.7)Imipenem 0(0)

Gentamicin 0(0)Levofloxacin 4(23.5)

Trimethoprim/sulfamethoxazole 15(88.2)Chloramphenicol 10(58.8)

Tetracycline 9(52.9)

Table 2 The incidence of resistance of different antibiotics amongdifferentisolates.

2017Vol.8 No.5:67



isolateswere found tobe resistant toAmpicillin andCefazolinwhile,E. coli and Klebsiellasp.werefoundtobehighlyresistantto Tobramycin, Cefoperazone, Amoxicillin-Clavulanic acid,Gentamycin, Cefepime and Amoxicillin (100% of the isolates).Moreover, all the Pseudomonas isolates were found to beresistanttoAmoxicillin-Clavulanicacid,Amoxicillin,TetracyclineandCefepime.

According to the data in Table 3 itwas found that, the testedisolates were found to be heterogeneous in their antibioticresistancepatternswhere;thetwelvetestedisolateshadresultedintendifferentpatterns.Themostrepresentedpatternswere(IB and II B), each representedby three isolates,while patterns(IA,IIAandIIE)wererepresentedbytwoisolatesandtheotherpatternswererepresentedbyoneisolate.TheresistantpatternsandtheMARindexoftheisolateswereconductedandthedatawererecordedinTable 3.

The antibacterial efficacy of Ag-NPs and ZnO-NPs against E. coli and K. pneumoniae isolates.The antibacterial efficacy of both tested nanoaprticles wasevaluated against twoE. coli and four K. pneumoniae isolates. Accordingtotheobtainedresults,itwasfoundthatalltheisolatesweresensitivetoAg-NPswithzoneof inhibitionwithdiameterranging between 14 and 15 mm except two K. pneumoniae isolates(K2andK4)thatwereresistant.Ontheotherhand,thesensitivityoftheisolatestoZnO-NPswerefoundtobelessthanthat to silver one; where only two isolates (E2 and K1) weresensitivetoZnO-NPswhilealltheotherisolatesdidnotshowanyinhibitionzonewithZnO-NPstreatmentasshowninTable 4. Generally, therewasnodifferencebetweenthediameterofinhibitionzonesobtainedby50and100µlofeitherAg-NPsorZnO-NPsFigure 7.

The efficacy of tested nanoparticles on enhancing the activity of different antibiotics In this experiment, the antibiotics to which the isolates werefound tobe resistantwere selected from the resistantpattern

of each isolateandtheantibacterialactivitiesoftheseantibioticsalonewerecomparedwith thosewith theircombinationswithAg-NPsorZnO-NPsusingdiscdiffusiontechnique.Theeffectofthese nanoparticles was determined as fold increasing in theinhibitionzonediameterandthedatawasrepresentedinTable 5.

According to the obtained results, it was clearly observedthat Ag-NPs not only have high antibacterial activity but alsothey show ability to enhance the activity of other antibioticsillustratedassynergisticeffectbyincreasingtheinhibitionzonediameteraroundtheantibioticdiscssupplementedwithAg-NPs.Moreover, the antibacterial activities of the tested antibioticsdiscscombinedwithAg-NPswerehigherthanthatofthesameantibioticswith ZnO-NPs asmost of antibiotics remained non-effectiveevenafterZnO-NPscombination(Figures 8 and 9).

The highest fold increase in the inhibition zone by Ag-NPscombination (8.878) was observed with Levofloxacin againstisolateE2 followedby thatofChloramphenicol (8.000) againstisolate K1. In addition, the highest fold increasewith ZnO-NPscombination was (1.939) with Amoxicillin/Clavulanic acid andTrimethoprim/SulfamethoxazoleagainstisolateE2.

The obtained inhibition zone diameters were interpreted tosensitive, intermediately resistant or resistant according toEUCASTClinicalBreakpoint[16].ThechangeinthesusceptibilityofeachisolatetoanyofthetestedantibioticswasdeterminedinthepresenceofeitherAg-NPsorZnO-NPsasshowninTable 5.

For the ease of comparison, Table 6wasconstructedtosummarizetheefficacyof testedcombinationsagainst the tested isolates.

Pattern Code Antimicrobial resistance pattern MAR

index

No. of isolates exhibit this

pattern

IA Am,AMC,CTX,C,TE

0.52

B Am,AMC,CTX,ATM,SXT 3

II

A Am,AMC,CTX,SXT,C,TE

0.6

2B Am,AMC,CTX,ATM,SXT,C 3C Am,AMC,CTX,LEV,SXT,C 1D Am,AMC,CTX,ATM,LEV,SXT 1E Am,AMC,CTX,ATM,SXT,TE 2

IIIA Am,AMC,CTX,LEV,SXT,C,TE

0.7

1B Am,AMC,CTX,ATM,LEV,SXT,TE 1C Am,AMC,CTX,ATM,SXT,C,TE 1

*Am: Ampicillin; AMC: Amoxicillin/clavulanic‏ acid; CTX: Cefotaxime;ATM:Aztreonam;IPM:Imipenem;GEN:Gentamicin;LEV:Levofloxacin;SXT: Trimethoprim/sulfamethoxazole; C: Chloramphinicol; TE:Tetracycline.

Table 3ResistancepatternsandMARindexoftestedisolates.

IsolatesCode

Mean of inhibition zone diameter (mm) ± SDAg-NPs ZnO-NPs

50μl 100μl 50μl 100μlE1 15±0.12 15±0.21 0±0.00 0±0.00E2 14±0.12 14±0.26 12±0.17 12±0.35K1 15±0.20 15±0.25 13±0.15 13±0.29K2 0±0.00 0±0.00 0±0.00 0±0.00K3 15±0.15 15±0.20 0±0.00 0±0.00K4 0±0.00 0±0.00 0±0.00 0±0.00

*E:E. coli;K:K. pneumonia;SD:Standarddeviation.Eachresultwerein triplicate.

Table 4 AntibacterialactivityofAg-NPsandZnO-NPsagainsttheselectedE. coli and K. pneumoniae isolates.

Figure 7 The antibacterial activity of Ag-NPs (A) and ZnO-NPs(B) against certain E. coli isolate;where,(1) and (2) are representativestotheinhibitionzonesobtainedat50and100μlofcorrespondingnanoparticles,respectively.

1

2017Vol.8 No.5:67



Figure 8 TheenhancedefficacyofAg-NPsontheantibacterialactivityofAztreonam(A)andAmoxicillin/clavulanicacid (B)oncertainE. coli isolate;where; theeffectofantibioticdiscaloneisontheleftandtheeffectofantibioticdiscsupplementedwithAg-NPsisontheright.

A B

Figure 9 The enhanced efficacy of ZnO-NPs on the antibacterial activity of Amoxicillin/clavulanic on one of K. pneumoniae isolateswhere;theeffectofantibioticdiscaloneontheleftandtheeffectoftheantibioticdiscsupplementedwithZnO-NPsontheright.

AccordingtotheobtaineditwasclearlydemonstratedthatAg-NPs are able to interestingly enhance the efficiency of someantibiotics.AmongsixisolatestestedwithAmpicillin,fourisolateshad been changed to intermediately resistant, one isolate had beenchangedtosensitivewhileonlyoneisolatestillresistant.Inaddition, for ;acidAmoxicillin/Clavulanic‏ two isolateshadbeenchanged to sensitive, two isolates two intermediately resistantand two isolates still resistant.Moreover, theTetracyclineandTrimethoprim/Sulfamethoxazole combination with Ag-NPs hadresulted in increasing the susceptibility of all tested isolatesto either sensitive or intermediately resistant. However, thecombination between Ag-NPs and other antibiotics such as

CefotaximeresultedinincreasingininhibitionzonediameterbutitstillintheresistantrangeindicatingthattheeffectofAg-NPscouldbevariedaccordingtoantibioticclassandmodeofaction.

ThesefindingagreedwiththatobtainedbyDengetal.[23]whostudied themechanism of synergistic activity between Ag-NPsand antibiotics (Ampicillin, Penicillin, Enoxacin, Kanamycin andTetracycline) on MDR Salmonella typhimurium DT 104. They reportedthatEnoxacin,kanamycin,NeomycinandTetracyclineshowed synergistic growth inhibition and antibiotic - nanocomplexes formationwas also detected.While, Ampicillin andPenicillin neither form complexes nor showed synergism with

2017Vol.8 No.5:67



Isolate code Tested antibiotic TreatmentType

Mean of inhibition zone diameter (mm) ± SD

Fold increase in inhibition zone diameter

Combination's susceptibility

K1

Am

Am only 0±0.00 - -

Am + Ag 15±0.58 3.592 I

Am+ ZnO 0±0.00 0.000 R

AMC

AMConly 0±0.00 - -

AMC+Ag 15±0.50 3.592 I

AMC+ZnO 13±0.50 2.449 R

CTX

CTXonly 9±0.29 - -

CTX+Ag 13±0.50 1.086 R

CTX+ZnO 12±0.00 0.778 R

C

Conly 0±0.00 - -

C+Ag 21±1.00 8.000 S

C+ZnO 0±0.00 0.000 R

TE

TE only 0±0.00 - -

TE + Ag 20±0.58 7.163 S

TE + ZnO 0±0.00 0.000 R

K2

Am

Am only 0±0.00 - -

Am + Ag 18±0.58 5.612 S

Am+ ZnO 0±0.00 0.000 R

AMC

AMConly 0±0.00 - -

AMC+Ag 17±1.00 4.898 I

AMC+ZnO 0±0.00 0.000 R

CTX

CTXonly 0±0.00 - -

CTX+Ag 20±0.89 7.163 R

CTX+ZnO 0±0.00 0.000 R

ATM

ATM only 0±0.00 - -

ATM + Ag 17±0.50 4.898 R

ATM + ZnO 0±0.00 0.000 R

SXT

SXT only 0±0.00 - -

SXT + Ag 17±0.00 4.898 S

SXT + ZnO 0±0.00 0.000 R

Table 5 Antimicrobial activity of antibiotics alone and in combinationwith Ag-NPs or ZnO-NPs against selected resistant E. coli and K. pneumoniae isolates.

2017Vol.8 No.5:67







E1

Am

Am only 0±0.00 - -

Am + Ag 15±0.50 3.592 I

Am+ ZnO 0±0.00 0.000 R

AMC

AMConly 0±0.00 - -

AMC+Ag 18±0.58 5.612 S

AMC+ZnO 11±0.50 1.469 R

CTX

CTXonly 0±0.00 - -

CTX+Ag 0±0.00 0.000 R

CTX+ZnO 0±0.00 0.000 R

ATM

ATM only 0±0.00 - -

ATM + Ag 15±0.00 3.592 R

ATM + ZnO 10±0.58 1.041 R

SXT

SXT only 0±0.00 - -

SXT + Ag 17±0.58 4.898 S

SXT + ZnO 0±0.00 0.000 R

TE

TE only 11±0.00 - -

TE + Ag 22±1.15 3.000 S

TE + ZnO 11±0.00 0.000 R

K3

Am

Am only 0±0.00 - -

Am + Ag 14±0.00 3.000 I

Am+ ZnO 0±0.00 0.000 R

AMC

AMConly 0±0.00 - -

AMC+Ag 12±0.76 1.939 R

AMC+ZnO 0±0.00 0.000 R

CTX

CTXonly 0±0.00 - -

CTX+Ag 0±0.00 0.000 R

CTX+ZnO 0±0.00 0.000 R

ATM

ATM only 9±0.00 - -

ATM + Ag 11±0.50 0.494 R

ATM + ZnO 10±0.00 0.235 R

SXT

SXT only 0±0.00 - -

SXT + Ag 15±0.50 3.592 I

SXT + ZnO 0±0.00 0.000 R

C

Conly 0±0.00 - -

C+Ag 14±0.29 3.000 I

C+ZnO 0±0.00 0.000 R

2017Vol.8 No.5:67







K4

Am

Am only 0±0.00 - -

Am + Ag 10±0.58 1.041 R

Am+ ZnO 0±0.00 0.000 R

AMC

AMConly 0±0.00 - -

AMC+Ag 0±0.00 0.000 R

AMC+ZnO 0±0.00 0.000 R

CTX

CTXonly 0±0.00 - -

CTX+Ag 14±0.58 3.000 R

CTX+ZnO 0±0.00 0.000 R

LEV

LEVonly 10±0.00 - -

LEV+Ag 19±0.50 2.610 S

LEV+ZnO 11±0.29 0.210 R

SXT

SXT only 0±0.00 - -

SXT+ Ag 14±0.00 3.000 I

SXT+ ZnO 0±0.00 0.000 R

C

Conly 0±0.00 - -

C+Ag 12±0.58 1.939 R

C+ZnO 0±0.00 0.000 R

TE

TE only 0±0.00 - -

TE + Ag 13±0.29 2.449 I

TE+ Zn 0±0.00 0.000 R

E2

Am

Am only 0±0.00 - -

Am + Ag 14±0.29 3.000 I

Am+ ZnO 0±0.00 0.000 R

AMC

AMConly 10±0.00 - -

AMC+Ag 19±1.15 1.984 S

AMC+ZnO 16±1.00 1.116 I

CTX

CTXonly 0±0.00 - -

CTX+Ag 13±0.00 2.448 R

CTX+ZnO 0±0.00 0.000 R

ATM

ATM only 9±0.00 - -

ATM + Ag 23±0.58 5.530 S

ATM + ZnO 9±0.00 0.000 R

LEV

LEVonly 0±0.00 - -

LEV+Ag 22±0.29 8.878 S

LEV+ZnO 10±0.29 1.041 R

SXT

SXT only 0±0.00 - -

SXT+ Ag 16±1.00 4.224 S

SXT+ ZnO 12±0.00 1.939 I

TE

TE only 0±0.00 - -

TE + Ag 12±0.29 1.939 I

TE + ZnO 0±0.00 0.000 R*E: E. coli; K: K. pneumonia; *Am: Ampicillin; AMC: Amoxicillin/clavulanic‏ acid; CTX: Cefotaxime; ATM: Aztreonam; LEV: levofloxacin; SXT:Trimethoprim/sulfamethoxazole; C: Chloramphenicol; TE: tetracycline; *R: resistant; I: intermediately resistant and S: sensitive; *SD: Standarddeviation.

2017Vol.8 No.5:67



Ag-NPs. They explained that the suggestedmechanism of thissynergismwithsomeantibioticsisthatfirstly,antibiotic−AgNPscomplex are formed then they interacts more strongly withthe bacterial cells and causes more Ag+release,thuscreatingatemporal high concentration of Ag+ near the bacteria cellwallthatleadstogrowthinhibitionofthebacteria.

Ourfindingsabout theenhancingactivityof silveragreedwiththat of Sindhu et al. [20]whocomparedtheactivityofGentamycinand Chloramphenicol antibiotics alone with that with Ag-NPs.Theyreportedthat theantibacterialactivityofbothantibioticsincreased in thepresenceofAg-NPsagainst thetestedstrains.Also, Kora and Rastogi [24] studied the effect of chemicallysynthesizedAg-NPscappedwiththreedifferentcappingagents;citrate,sodiumdodecylsulfate(SDS)andpolyvinylpyrrolidone(PVP) on the antibacterial activity of Streptomycin, Ampicillin,and Tetracycline against E. coli and S. aureus. They reported thatthehighestpercentageofenhancement inactivityagainstE. coliwasobtained incombinationofAmpicillinwithPVPandSDScappednanoparticles;while,StreptomycincombinationwithPVPcappednanoparticlesshowedthehighestactivityagainst S. aureus.

Furthermore, the observed lower activities of ZnO-NPs inour study agree with the findings of Meruvu et al. [25] whoinvestigated theantibacterialactivityofZnO-NPscombinationswithGentamycin,NitrofurantoinandCiprofloxacinagainstE. coli where,theresultsshowedthattheactivityofnanoparticlesalonewaslowerthanthatofantibiotics alonethanthatofantibiotics-ZnO-NPscombination.

ConclusionTheincreasingantimicrobialresistanceamongbacterial strains is agreatthreatthatneedsrapidandseriousactionstobetaken.Due to their unique antibacterial activity; silver nanoparticlescouldbeevaluatedasalternativestothetraditionalantibioticsorusedtoenhancetheefficacyofcertainantibiotics.

Acknowledgment AuthorsthankDr.ShereenF.Gheida,DepartmentofDermatology andVenereology,FacultyofMedicine,TantaUniversity,Egypt,Whohavehelpedincollectingsamplesfrompatient.

Antibiotic No. of tested isolates Antibiotic-Nano combination

No. (%) of isolates changed to intermediately sensitive

No. (%) of isolates changed to sensitive

Am 6Amp–Ag-NPs 4(66.7) 1(16.6)Amp–ZnO-NPs 0(0) 0(0)

AMC 6AMC–Ag-NPs 2(33.3) 2(33.3)AMC–ZnO-NPs 0(0) 0(0)

CTX 6CTX–Ag-NPs 0(0) 0(0)CTX–ZnO-NPs 0(0) 0(0)

C 3C–Ag-NPs 1(33.3) 1(33.3)C–ZnO-NPs 0(0) 0(0)

TE 4TE–Ag-NPs 2(50) 2(50)TE–ZnO-NPs 0(0) 0(0)

ATM 4ATM–Ag-NPs 0(0) 1(25)ATM–ZnO-NPs 0(0) 0(0)

SXT 5SXT–Ag-NPs 2(40) 3(60)SXT–ZnO-NPs 0(0) 0(0)

LEV 2LEV–Ag-NPs 0(0) 2(100)LEV–ZnO-NPs 0(0) 0(0)

*Am:Ampicillin;AMC: /TrimethoprimSXT:Aztreonam;ATM:Tetracycline;TE:Chloramphenicol;C:Cefotaxime;CTX:acid;Amoxicillin/clavulanic‏sulfamethoxazole;LEV:levofloxacin.

Table 6Summaryoftheefficacyofdifferentantibiotics-nanoparticlescombinationsonthesusceptibilityoftestedisolates.

References1 HealthPolicyBrief(2015)AntibioticResistance.HealthAffairs12:1-7.

2 WorldHealthOrganization: Globaltuberculosisreport11:152-191.

3 Melakea NA, Eissaa NA, Keshkb TF, Sleema AS (2015) Prevalenceof multidrug-resistant bacteria isolated from patients with burninfection.MenoufiaMedJ28:677-684.

4 SilverLL(2011)Challengesofantibacterialdiscovery.ClinMicrobiolRev24:71-109.

5 NaqviSZH,KiranU,AliMI,JamalA,HameedA,etal.(2013)Combined

efficacyofbiologicallysynthesizedsilvernanoparticlesanddifferentantibioticsagainstmultidrug-resistantbacteria.IntJNanomedicine8:3187-3195.

6 Singh R, Nawale LU, Arkile M, Shedbalkar UU, Wadhwani SA,et al. (2015) Chemical and biological metal nanoparticles asantimycobacterialagents:Acomparativestudy. I JAntiMicAg46:183-188.

7 RashidBZ,OmarRA,SalihSJ(2016)Characterizationandantimicrobialefficiencyofsilvernanoparticlesbasedreductionmethod.IntJCurrMicrobiolAppSci5:802-810.

2017Vol.8 No.5:67



8 HoltJG,KriegNR,SneathPHA,StaleyJT,WilliamsST(1994)GenusAcetobacter and Gluconobacter.Bergey’smanualofdeterminativebacteriology. 9th edn. Williams and Wilkens,MDUSA14:668-675.

9 FangJ,ZhangC,MuR(2005)ThestudyofdepositedsilverparticulatefilmsbysimplemethodforefficientSERS.ChemPhysLett 401:271-275.

10 ZawrahMF, Abd El-Moez SI (2011) Antimicrobial activities of goldnanoparticlesagainstmajorfoodbornepathogens.LifesciJ8:37-44.

11 Šileikaitė A, Prosyčevas I, Puišo J, Juraitis A, Guobienė A (2006)Analysisofsilvernanoparticlesproducedbychemical reductionofsilversaltSolution.MaterSciMedzg12:287-291.

12 Salahuddin NA, El-Kemary M, Ibrahim EM (2015) Synthesis andcharacterizationofZnOnanoparticlesviaprecipitationmethod:Effectofannealingtemperatureonparticlesize.JNanosciNanotechnol5:82-88.

13 MieG(1908)Contributionstotheopticsofturbidmedia,especiallycolloidalmetalsolutions.Ann.Phys25:377-445.

14 Vijayakumara M, Priyab K, Nancy FT, Noorlidaha A, Ahmed ABA(2013) Biosynthesis, characterisation and anti-bacterial effect ofplant-mediated silver nanoparticles usingArtemisia nilagirica. IndCropPro41:235-240.

15 Bauer AW, Kirby WMM, Sherris JC, Truck M (1966) Antibioticsusceptibilitytestingbyastandardizeddiskmethod.AmerJClinPath45:493-496.

16 European Committee for Antimicrobial Susceptibility Testing(EUCAST)(2012)Antimicrobialactivity22.

17 OlayinkaA,OlayinkaB,OnileB (2009)AntibioticsusceptibilityandplasmidpatternofPseudomonas aeruginosa from the surgical unit ofaUniversityteachinghospitalinnorthcentralNigeria.IntJMedSci1:79-83.

18 Jayaraman S,ManoharanM,IllanchezianS,SekherR,SathyamurthyS (2010) Plasmid analysis and prevalence of multidrug resistantStaphylococcus aureus reservoirs in Chennai city, India. Asian JPharmacolLifeSci10:117-125.

19 Lee SM, Song KC, Lee BS (2010) Antibacterial activity of silvernanoparticlespreparedbya chemical reductionmethod.Korean JChemEng27:688-692.

20 SindhuPD,MukherjeeA,ChandrasekaranN(2013)Synergisticeffectof biogenic silver nanocolloid in combination with antibiotics: Apotenttherapeuticagent.IntJPharmPharmSci5:292-295.

21 ChiengBW,LooYY(2012)SynthesisofZnOnanoparticlesbymodifiedpolyolmethod.MaterLett73:78-82.

22 Abbas HA, El-Masry EM, Shaker GH, Mohsen I (2013) BacterialetiologyandantimicrobialresistanceofburnwoundinfectionsinaburnunitinHehiageneralhospitalinEgypt.IntJBiolPharmRes4:1251-1255.

23 Deng H, McShan D, Zhang Y, Sinha SS, Arslan Z, et al. (2016)Mechanistic study of the synergistic antibacterial activity ofcombinedsilvernanoparticlesandcommonantibiotics.EnvironSciTechnol50:8840-8848.

24 KoraAJ, Rastogi L (2013) Enhancement of antibacterial activity ofcapped silver nanoparticles in combination with antibiotics, onmodel Gram-negative and Gram-positive bacteria. Bioinorg ChemAppl8:319-355.

25 Meruvu H, Vangalapati M, Chippada SC, Rao Bammidi SR (2011)Synthesis and characterization of zinc oxide nanoparticles and itsantimicrobial activity against Bacillus subtilis and Escherichia coli. Rasayan JChem4:217-222.

Documents

Investigation of the Efficacy of Synthesized Silver and Zinc … · Investigation of the ... antibacterialactivity of Ag-NPs and ZnO-NPs against bacterial pathogens, no available