Antimicrobial susceptibility of Brazilian Clostridium difficile strains determined by agar dilution and disk diffusion

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Antimicrobial

susceptibility

of

Brazilian

Clostridium

difficile

strains

determined

by

agar

dilution

and

disk

diffusion

Edmir

Geraldo

Fraga

_,

_Antonio

_Carlos

_Nicodemo

,

Jorge

Luiz

Mello

Sampaio

c_{UniversidadedeSãoPaulo(USP),FaculdadedeFarmácia,SãoPaulo,SP,Brazil}

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o

Articlehistory:

Received26January2016 Accepted3July2016

Availableonline16August2016 Keywords: Clostridiumdifficile Diskdiffusion Agardilution Susceptibilitytesting

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Clostridiumdifficileisaleadingcauseofdiarrheainhospitalizedpatientsworldwide.While metronidazoleandvancomycinarethemostprescribedantibioticsforthetreatmentofthis infection,teicoplanin,tigecyclineandnitazoxanidearealternativesdrugs.Knowledgeon theantibioticsusceptibilityprofilesisabasicsteptodifferentiaterecurrencefrom treat-mentfailureduetoantimicrobialresistance.BecauseC.difficileantimicrobialsusceptibility islargelyunknowninBrazil,weaimedtodeterminetheprofileofC.difficilestrainscultivated fromstoolsamplesofinpatientswithdiarrheaandapositivetoxinA/Btestusingbothagar dilutionanddiskdiffusionmethods.All50strainstestedweresensitivetometronidazole accordingtoCLSIandEUCASTbreakpointswithanMIC90valueof2␮g/mL.Nitazoxanide

andtigecyclinewerehighlyactiveinvitroagainst thesestrainswith anMIC90valueof

0.125␮g/mLforbothantimicrobials.TheMIC90were4␮g/mLand2␮g/mLforvancomycin

andteicoplanin,respectively.Aresistancerateof8%wasobservedformoxifloxacin.Disk diffusioncanbeusedasanalternativetoscreenformoxifloxacinresistance,nitazoxanide, tigecyclineandmetronidazolesusceptibility,butitcannotbeusedfortesting glycopep-tides.OurresultssuggestthatC.difficilestrainsfromSãoPaulocity,Brazil,aresusceptible tometronidazoleandhavelowMIC90valuesformostofthecurrenttherapeuticoptions

availableinBrazil.

©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction

Clostridiumdifficileisaspore-formingGram-positivebacillus. Thismicroorganismproducestwomajortoxins,enterotoxinA

∗ _{Correspondingauthors.}

E-mailaddresses:acnicodemo@uol.com.br(A.C.Nicodemo),sampaio@usp.br(J.L.Sampaio).

andcytotoxinB,thatcancausediarrhea,pseudomembranous colitis,colondilation,sepsis,andevendeath.1

TheincidenceandseverityofC.difficileinfections(CDI)is growinginmanycountriesdueinparttothedisseminationof

http://dx.doi.org/10.1016/j.bjid.2016.07.004

1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ahypervirulentstrainknownasNorthAmericaPulsetype1 (NAP1)orribotype027.2

Until the 1980s, there was little interest in research-ing newantibioticsforthe treatmentofCDI because most patientsrespondedwelltotreatmentwithmetronidazoleor oralvancomycin.Morerecently,infectionrecurrenceandthe limitationsoftheavailabletherapeuticoptionshavebecome clearer.3_{Therearestilldoubtsabouttheaccuracyand}

corre-lationofdifferentmethodsusedtoevaluateinvitroantibiotic sensitivityaswellasthesensitivityofC.difficilestrainstothe recommendedtreatmentregimens.

Thisstudyassessedthesusceptibilityprofilesofa collec-tionofC.difficilestrainscultivatedfromstoolsofinpatients withdiarrheainsixtertiaryhospitalsinSãoPaulo,Brazil.We alsoaimedtoevaluateifdiskdiffusionmethodcouldbean alternativeforsusceptibilitytestingofthemaindrugsused inthetreatmentofCDIs.Itwasnotapurposeofthestudyto evaluatetheepidemiologicaldataofthepatients.

Materials

and

methods

Clostridiumdifficilestrains

ConsecutiveclinicalstrainsofClostridiumdifficile(n=50)were cultivatedfromstoolsamplesofinpatientswithdiarrheain sixtertiary hospitalsinSãoPaulofromMarchtoDecember 2013(onesampleperpatient).Stoolsampleswererandomly selected for culture based on their positivity when tested withtheProScpectTM _{C. difficileToxinA/BMicroplateAssay} (ThermoScientific).StoolculturesforC.difficilewerecarried out as previously described, with modifications.4 _In

sum-mary,approximately 0.5goffeceswere mixed with0.5mL 95% ethanol,vortexed and incubated at roomtemperature (20–25◦_{C) for 1h. The suspension was vortxed again and} twodrops wereplated onBrucella agar supplementedwith 5%horseblood and 0.2%sodiumtaurocholate. Plateswere incubatedina2.5Lanaerobic jarcontaingtheAtmosphere GenerationSystemAnaeroGen(Oxoid-ThermoScientific)for 72hat36◦_{C.Identificationtothespecieslevelwasachievedby} MALDI-ToFMSusingtheMALDIBiotyperLTSystem(Bruker).

Thestrainswere storedin10%skimmilkat−70◦Cand subculturedonBrucellaagarwith5%horsebloodtwicebefore utilizationinsusceptibilitytests.

Antimicrobialsusceptibilitytesting

The antimicrobials tested in this study were: metronida-zole(Sigma-Aldrich),moxifloxacin (Sigma-Aldrich), nitazox-anide (Farmoquímica), teicoplanin (Sigma-Aldrich), tigecy-cline(Pfizer),andvancomycin(Sigma-Aldrich).

Diskdiffusion was performedas described by Erikstrup etal.5_{Culturedstrainsweresuspendedinthioglycollatebroth}

toa density of1.0 McFarland (≈3.0×108CFU/mL) withthe aidofDensiCheck® _{(bioMérieux).Thesuspensionwasthen} seededontoBrucellaBloodAgarsupplementedwith10%sterile defibrinatedlysedhorseblood,hemin(5␮g/mL)andvitamin K(1␮g/mL).TooptimizethegrowthofC.difficile,plateswere pre-reducedfor24hinananaerobic atmospheregenerated bythe AnaeroGensystem (Oxoid-ThermoScientific) before

use.Forinoculumpreparation,inoculationandincubationthe 15-15-15rulewasfollowed.5

After24hofincubationat36◦_{Cinanaerobicatmosphere,} generatedwiththe aidoftheAnaeroGenAtmosphere Gen-eration Systems (Oxoid-Thermo Scientific), inhibition zone diameters weremeasured underreflectedlightconsidering 100% inhibition. Duplicate tests were performed for each strainontwoseparatedays.Theinhibitionzonediameters werecorrelatedwiththeminimuminhibitoryconcentrations (MICs)obtainedbyagardilutionforeachstrainanddrug com-bination.

For nitazoxanide and metronidazole 6-mm paper disks were prepared by adding 10␮L of a 0.5mg/mL solution in dimethyl sulfoxide (Sigma), while for vancomycin and teicoplanin6-mmpaperdiskswithapotencyof5␮gwere pre-paredbyadding10␮Lofa0.5mg/mLsolutioninreagentgrade water.A30␮gteicoplanindisk(Oxoid-ThermoScientific)was alsotested.Fortigecyclineandmoxifloxacin,weused com-merciallyavailabledisks(Oxoid-ThermoScientific)containing 15and5␮g,respectively.Therearecurrentlynointerpretative criteriafordiskdiffusionwhentestingC.difficile.

For agar dilution bacterialstrainswere tested according to the Clinical and Laboratory Standards Institute (CLSI)6

and theEuropeanCommitteeonAntimicrobial Susceptibil-ityTesting(EUCAST)7_{guidelines.Bacterialsuspensionswere}

prepared in thioglycollatebroth to 0.5McFarland turbidity (≈1.5×108CFU/mL)before1␮Lofeachsuspensionwas trans-ferredtotheagar plateswiththeaidofaSteersreplicator. Platescontainingantibioticwerestampedstartingfromthe lowest concentration. The MIC was defined as the lowest antibioticconcentrationinhibitingvisiblegrowthafter48hof incubationat37◦_{Cinanaerobiosis.Testswereperformedin} duplicates.

TheCLSIbreakpointsforMICswereusedfor metronida-zole and moxifloxacin,8 _{while EUCAST criteria were used}

formetronidazoleandvancomycin.7_{Theinterpretative}

crite-ria are summarized in Table 2. There are currently no interpretative criteria fortigecycline, teicoplanin,and nita-zoxanide;consequently, onlyMIC50 and MIC90 valueswere calculated.Forteicoplaninandnitazoxanide,theECOFFinder spreadsheet9 _{was used to estimate epidemiological cut-off}

values(ECOFFs).C.difficileATCC700057strainwasusedfor qualitycontrolandtestedsimultaneouslywitheachbatchof antimicrobialsusceptibilitytests.

Statisticalanalysis

Formetronidazole,moxifloxacin,andvancomycincategorical agreement between agar dilution and tentative interpreta-tivecriteriafordiskdiffusionwasevaluatedusingCLSIand EUCASTbreakpointsforagardilution.Theerrorswere classi-fiedaspreviouslydescribed.10

Results

From March 1st to December 31st 2013 there were 1884 patientsforwhichthedetectionofC.difficiletoxinsAandBby ELISAwasorderedbytheattendingphysicians.Atotalof239 (12.7%)patientshadapositivetest.Themeanageofpatients

Table1–Minimalinhibitoryconcentrationsand inhibitionzonediametersobtainedforC.difficileATCC 700057bytheagardilutionanddiskdiffusionmethods.

Antimicrobial Agardilution(␮g/mL) Diskdiffusion(mm)

Metronidazole 0.25–0.5 38–42 Moxifloxacin 0.5–4 22–25 Nitazoxanide 0.06–0.125 26–30 Teicoplanin 0.25–2 15–20a 24–28b Tigecycline 0.06–0.125 35–40 Vancomycin 0.25–2 21–25 a _{5␮gteicoplanindisk.} b _{30␮gteicoplanindisk.}

withapositivetestresultwas61.3yearsandmost(61.1%)were female.Atotalof50fecalsampleswereramdomlyselected andculturedforisolationofC.difficile.Therecoveryratewas 100%.TheseisolateswereconfirmedtobeC.difficilebymass spectrometryandwereusedforsusceptibilitytests.

AlltheMICresultsforthequalitycontrolstrainC.difficile ATCC700057wereinsidetheexpectedrangerecommendedby theCLSI(Table1).Forteicoplanin,forwhichthereisnoCLSI orEUCASTexpectedrangeforthisstrain,theMICrangewas 0.25–2␮g/mLandtheinhibitionzonediameterrangedfrom 15to20mmand24 to28mmforthe 5␮gand 30␮gdisks, respectively(Table1).

Alltestedstrainsweresusceptibletometronidazolebothby CLSIandEUCASTcriteria.TheMIC50andMIC90valuesare dis-playedinTable2.Using30mmasthesusceptibilitybreakpoint fordiskdiffusiontherewould be100%categoryagreement betweenagardilutionanddiskdiffusionmethods(Fig.1A).

TherearecurrentlynoCLSIbreakpointsforglycopeptides whentestingC.difficile.Twenty-onestrains(42%)hadanMIC ≤2␮g/mLforvancomycinandwereclassifiedassusceptible usingtheEUCASTbreakpointforC.difficile.MIC50andMIC90 valuesaredisplayedinTable2.

Using the 2␮g/mLEUCAST susceptibility breakpoint for vancomycinnoneofthevaluesobtainedforinhibitionzone diameterwouldresultinamajorerrorrateunder1.5%(Fig.1B). Thesamewasobservedforteicoplanin(Fig.1CandD),ifwe usethesameEUCASTsusceptibilitybreakpointavailablefor vancomycin,bothwiththehe5␮gorthe30␮gdisks.

Fornitazoxanide,theinterpretationcriteriaforDDandMIC havenotbeendefinedbyCLSIorEUCAST.TheMIC50andMIC90 valuesaredisplayedinTable2.Allstrainswithaninhibition

zone diameter≥24mmhadan MICvalue≤0.25␮g/mL, the calculatedECOFFvalue(Fig.1E).

There are currently no tigecycline susceptibility break-pointsaccordingtoCLSIorEUCAST.TheMIC50andMIC90are displayedinTable2.ThecalculatedECOFFwas0.25␮g/mL.All isolates,exceptone,hadaninhibitionzonediameter≥30mm andanMIChigherthan0.25␮g/mL(Fig.1F).

Formoxifloxacin,boththeMIC50andMIC90were4␮g/mL (Table2).Four(8%)strainshadhighMICvaluesof32␮g/mL (Fig. 1G). Applying the CLSI susceptibility breakpoint of 2␮g/mLformoxifloxacin,only6%ofthestrainswouldbe con-sideredsusceptible.Using thisMICbreakpoint,noneofthe valuesobtainedforinhibitionzonediameterwouldresultin averymajorerrorrateunder1.5%.

Discussion

Weevaluatedtheantimicrobialsusceptibilityprofileofa col-lectionof50C.difficilestrainsusingbothagardilutionanddisk diffusion.

All strains were susceptible to metronidazole, and the highestMICvaluewas2␮g/mL,twodilutionsbellowthe sus-ceptibilitybreakpointof8␮g/mLrecommendedbytheCLSI. Thesefindingsagreewitharecentpublicationthatevaluated alargecollectionofstrainsfromEuropeandfoundthesame MIC90values.11

Althoughwedidnotdetectresistantstrains,metronidazole resistanceinC.difficilehasbeenreportedveryrarely.Freeman et al.11_{foundonlyoneisolatewithanMICvalue≥8␮g/mL}

among916testedbyagardilution.Concerningthe metronida-zolediskdiffusionmethod5,5,12 _{wefoundthataninhibition}

zonediameter≥30mmforametronidazolediskcontaining 5␮gcanbeindicativeofsusceptibility, whileErikstrupand colleagues5_{recommendusing23mmasthebreakpoint.This}

differencebetweenbreakpointsismostprobablyduetothe numberofstrainstestedinthiswork,butallstrains classi-fied assusceptiblebythe 30mmbreakpointwould alsobe classifiedassusceptiblewiththe23mmbreakpoint.

Oralmetronidazolehasbeenrecommendedasthe treat-ment ofchoice for mild disease while oral vancomycin is usuallyrecommendedforthetreatmentofsevereinfections and recurrences.13 _{In this study the MIC}

90 for vancomycin was 4␮g/mL, which isone dilution above the valuefound by Freeman et al.11 _{Our resultsmay not becomparableto}

theirs,becausetheyusedtheWilkins-Chalgrenagarandwe

Table2–Susceptibilityprofileof50Clostridiumdifficileisolatesbyagardilution.

Antimicrobial MIC50

(␮g/mL)

MIC90 (␮g/mL)

Susceptible(%) Resistant(%) Susceptibility

breakpoint(␮g/mL)

Concentration

rangetested

(␮g/mL)

CLSI EUCAST CLSI EUCAST CLSI EUCAST

Metronidazole 1 2 100 100 0 0 ≤8 ≤2 0.25–32 Moxifloxacin 4 4 6 – 8 – ≤2 – 0.25–32 Nitazoxanide 0.06 0.12 – – – – – – 0.03–4 Teicoplanin 2 2 – – – – – – 0.25–32 Tigecycline 0.12 0.12 – – – – – – 0.03–16 Vancomycin 4 4 – 42 – 58 – ≤2 0.25–32

A

C

E

F

G

D

B

Inhibition zone diameter (mm)

Inhibition zone diameter - 5 µg disk (mm)

Inhibition zone diameter (mm) Inhibition zone diameter (mm)

Inhibition zone diameter (mm)

Inhibition zone diameter - 30 µg disk (mm) Inhibition zone diameter (mm)

Teicoplanin Nitazoxanide Moxifloxacin Tigecycline Teicoplanin Vancomycin MIC ( µ g/mL) MIC ( µ g/mL) MIC ( µ g/mL) MIC ( µ g/mL) MIC ( µ g/mL) MIC ( µ g/mL) MIC ( µ g/mL) 16 8 4 2 1 1 1 1 1 1 3 1 2 1 1 2 4 1 2 1 1 1 1 2 9 5 3 2 5 8 5 3 1 1 1 5 5 5 3 2 3 1 5 4 2 1 3 1 8 1 2 9 8 5 1 3 2 1 1 9 1 4 3 4 2 2 1 1 2 1 1 1 1 1 3 5 4 3 8 4 6 2 1 1 4 7 1 4 2 5 1 1 1 1 1 2 2 2 6 5 4 6 8 3 5 7 8 8 9 8 2 1 1 1 2 1 1 1 ≤ 8 µg/mL (CLSI) ≤ 2 µg/mL (EUCAST) ECOFF 95% 0.25 µg/mL ECOFF 0.25 µg/mL (EUCAST) ECOFF 4µg/mL (EUCAST) ≤ 2 µg/mL (EUCAST) 1 4 0.5 0.25 30 31 32 33 34 35 36 37 38 39 40 21 22 23 24 25 26 32 16 8 4 2 1 0.5 0.25 32 16 8 4 2 1 0.5 0.25 32 16 8 4 2 1 0.5 0.25 14 24 25 26 27 28 29 30 31 32 33 6 13 14 15 16 17 18 19 20 21 22 23 24 25 30 31 32 33 34 35 36 37 38 39 40 41 15 16 17 18 19 20 21 18 19 20 21 22 23 24 25 26 27 4 2 1 0.5 0.25 0.12 0.06 0.03 4 8 2 1 0.5 0.25 0.12 0.06 0.03 32 16 8 4 2 1 0.5 0.25 ≤ 2 µg/mL (CLSI)

Fig.1–Scattergramscomparinginhibitionzonediameterswithminimalinhibitoryconcentrationsobtainedbyagar dilutionfor50C.difficilestrains.

usedBrucellaagar.Bainesetal.14_{demonstratedthatMIC}

val-uesobtainedusingBrucellaagarforagardilutionwerelower thanthose obtainedwhen usingEteststrips.Erikstrupand colleagues5 _{used the Etest and notthe gold standard agar}

dilution. Consequently, this may explain why we did not findgoodcorrelation betweendisk diffusionandagar dilu-tion methods.Our resultscontrast tothose from Erikstrup andcolleagues,5 _{sinceinthis work noinhibitionzone size}

wouldresultinaverymajorerrorratebelow1.5%.Ourresults agreewellwithpreviousfindingsthatsupportedthewitdrawal oftheinterpretative criteriaforvancomycin diskdiffusion, whentestingStaphylococcus,fromCLSIdocuments.15_Thereare

noCLSIinterpretative criteriaforvancomycinwhentesting

C.difficile,whileEUCASTrecommendsasusceptibility break-pointof2␮g/mL.Basedonthisbreakpoint,Freemanetal.11

reported alow(0.87%)resistancerate whentesting alarge collection ofstrains. Although the results from this study are different from those of Freeman et al.,11 _{our findings}

are supportedbythe factthat themean vancomycin MICs obtainedforthequalitycontrolstrainC.difficileATCC700057 was2␮g/mL,whichisonedilutionbelowtheupperlimit.8

Teicoplaninand nitazoxanidearealternative treatments forCDI3,16_{buttherearecurrentlynosusceptibilitybreakpoints}

definedbyEUCASTorCLSI.TheMIC90obtainedforteicoplanin inthisstudyistwodilutionsabovethatobtainedbyBarbut et al.17 _{This differenceis probablydue tothe factthat we}

usedBrucellaagarandtheyusedWilkins-Chalgrenagar.Baines etal.14_{demonstratedthatagardilutionwithWilkins-Chalgren}

agargeneratesresultsonedilutionlowerthanthosegenerated byBrucellaagar.

InthisstudytheMIC90fornitazoxanidewas0.125␮g/mL. ThisfindingagreeswithapreviousstudyfromFreemanetal.18

that tested strains from ribotypes 001, 027 and 106 with reducedsusceptibilitytometronidazole.Todatethereareno reportsonthenitazoxanidediskdiffusionmethodforC. diffi-cile.Althoughinthisworkwedidnotfindnitazoxanideisolates withhighMICs,ourresultsindicatethat24mmcouldbeused asascreeningforisolateswithMICs≤0.125␮g/mL.

There are some reports in the literature that describe tigecyclineasanalternativetherapeuticoptionforpatients withrefractoryCDI.19_{InthisstudyweobtainedanMIC}

90of

0.125␮g/mLforthisantimicrobial.Thisvalueisthesameas obtainedbyHechtetal.20_{TherearecurrentlynoEUCASTor}

CLSIsusceptibility breakpointsfortigecyclinewhentesting C.difficile,butEUCASTrecommendsanECOFFof0.25␮g/mL. Using this value asa susceptibility breakpoint, 98% ofthe strainswouldbeconsideredsusceptible.Therearecurrently noreportsondiskdiffusionfortigecyclinewhentestingC. difficile.Althoughinthisworkwedidnotfindisolateswitha hightigecyclineMICs,ourresultsindicatethat30mmcould beusedasascreeningforisolateswithMICs≤0.5␮g/mL.

Resistance to fluoroquinolones has been suggested as a screening for the presence of the hyper virulent strain NAP1/BI/027.21,22 _{CLSIrecommends2␮g/mLasthe}

suscepti-bilitybreakpointvalue,whilestrainswithanMICof4␮g/mL shouldbeclassifiedasintermediate.EUCASThasno suscepti-bilitybreakpointformoxifloxacinwhentestingC.difficile,but recommendsanECOFFvalueof4␮g/mL.

InthisstudytheMIC90was4␮g/mLandfourstrainshad anMIC≥32␮g/mL.UsingCLSIbreakpoints6%ofthestrains wouldbeclassifiedassusceptible,86%intermediate,and8% resistant.Thisresistancerateismuchlowerthanthatfound inEurope(39.9%)byFreemanandcolleagues.11_Todatethere

isnoreportonthepresenceofthe027ribotypeemergingin Brazilandpossiblythislowresistancerateindicatesthe pre-dominanceofribotypesother than 027,but ribotypingwas notperformed inthis study.If we had used13mm asthe resistancebreakpoint,thenthefourresistantstrainswould havebeendetected.Wefoundasuperpositionof intermedi-ateandsusceptiblecategoriesbydiskdiffusion.Consequently, thismethodcannotbeusedtopreviewsusceptibilitybutcan beusedtoscreenformoxifloxacinresistance.

Conclusions

Allstrainstestedinthisstudyweresusceptibleto metronida-zoleaccordingtoEUCASTandCLSIbreakpoints.Nitazoxanide and tigecycline were higly active, both with an MIC90 of 0.125␮g/mL.TheMIC90 were 4␮g/mLand2␮g/mLfor van-comycinandteicoplanin,respectively.

The disk diffusion method can be used to screen for metronidazole, tigecycline and nitazoxanide susceptibility, andmoxifloxacin resistancebutcannotbeusedfortesting vancomycinandteicoplanin.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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