eucast detection of resistance mechanisms 170711

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EUCASTguidelinesfordetectionofresistancemechanismsandspecificresistancesofclinicaland/or

epidemiologicalimportance

Version2.01July2017

1 Basedonversion1.0fromDecember2013bytheEUCASTsubcommitteefordetectionofresistancemechanismsand specific resistances of clinical and/or epidemiological importance. Authors of the original version areacknowledged: ChristianG.Giske (Sweden, EUCAST and EARS-Net CoordinationGroup; chairman), LuisMartinez-Martinez(Spain),RafaelCantón(Spain,EUCAST),StefaniaStefani(Italy),RobertSkov(Germany),YouriGlupczynski(Belgium), Patrice Nordmann (France), Mandy Wootton (UK), Vivi Miriagou (Greece), Gunnar Skov Simonsen(Norway,EARS-NetCoordinationGroup),HelenaZemlickova(CzechRepublic,EARS-NetCoordinationGroup),JamesCohen-Stuart(TheNetherlands),andMarekGniadkowski(Poland).

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Contents

Section Page 1.Introduction 32.Carbapenemase-producingEnterobacteriaceae 43.Extended-spectrumβ-lactamase-producingEnterobacteriaceae 134.AcquiredAmpCβ-lactamase-producingEnterobacteriaceae 225.PolymyxinresistanceinGram-negativebacilli 266.CarbapenemresistanceinP.aeruginosaandAcinetobacter 287.MethicillinresistantStaphylococcusaureus 308.Glycopeptidenon-susceptibleStaphylococcusaureus 339.VancomycinresistantEnterococcusfaeciumandEnterococcusfaecalis 3710.Penicillinnon-wild-typeStreptococcuspneumoniae 42

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1.IntroductionThecurrentdocumentisanupdateoftheguidelinesdevelopedbytheEUCASTsubcommitteeondetectionofresistancemechanisms.TheEUCASTSteeringCommitteehascarriedoutthecurrentupdate.Thedocumenthasbeendevelopedmainlyforroutineuseinclinicallaboratoriesanddoesnotcovertechnicalproceduresforidentificationofresistancemechanismsatamolecularlevelbyreference or expert laboratories. However, much of the content is also applicable to nationalreference laboratories. Furthermore, it is important to note that the document does not coverscreeningforasymptomaticcarriage(colonization)ofmultidrug-resistantmicroorganismsordirectdetectionofresistanceinclinicalsamples.All chapters in this document contain a definition of the mechanism or specific resistance, anexplanationof theclinicaland/orpublichealthneed fordetectionof themechanismor specificresistance, an outline description of recommended methods of detection, and references todetaileddescriptionsofthemethods.Theneedforidentificationofresistancemechanismandthelevel of identification needed for public health or infection control purposes may vary bothgeographically and temporally depending on the prevalence and heterogeneity of differentresistancemechanisms. The guidelines have been developed by conducting literature searches,and recommendations arebasedonmulti-center studiesormore thanone single center study.Severalmethodscurrentlyunderdevelopmenthavenotbeenincludedintheguidelinesasmulti-centerevaluationsormultiplesinglecenterevaluationsareyettobecompleted.DraftversionsoftheseguidelinesweresubjecttowideconsultationthroughEUCASTconsultationcontactlists,theEUCASTwebsiteandECDCfocalpointcontacts.Wehaveas faraspossibleusedgenerictermsfortheproductspresented inthedocument,butexcludingallspecificproductnameswouldhavemadesomeoftherecommendationsunclear.Itshouldbenoted that someresistancemechanismsdonotalwaysconferclinical resistance.Thiscouldbeduetomechanismsnotbeingexpressedorexpressedonlyatalow-level,whichwillnotgiverisetophenotypicresistance.Hence,whiledetectionofthesemechanismsmayberelevantforinfectioncontrolandpublichealth,itmaynotbenecessaryforclinicalpurposes.Consequently,for some mechanisms, particularly extended-spectrum β-lactamases and carbapenemases inGram-negative bacilli, detection of the mechanism does not in itself lead to classification asclinicallyresistant.ChristianG.GiskeChairmanofEUCAST,PastChairmanoftheSubcommitteeondetectionofresistancemechanisms

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2. Carbapenemase-producingEnterobacteriaceaeImportanceofdetectionofresistancemechanismRequiredforclinicalantimicrobialsusceptibilitycategorization NoInfectioncontrolpurposes YesPublichealthpurposes Yes

2.1DefinitionCarbapenemasesareβ-lactamasesthathydrolyzepenicillins,inmostcasescephalosporins,andtovarious degrees carbapenems and monobactams (the latter are not hydrolyzed by metallo-β-lactamases).2.2Clinicaland/orepidemiologicalimportanceThe problemof dissemination of carbapenemases in Europe dates from the second half of the1990sinseveralMediterraneancountries,andwasobservedmainlyinPseudomonasaeruginosa(1).Intheearly2000s,GreeceexperiencedanepidemicoftheVeronaintegron-encodedmetallo-β-lactamase (VIM) among Klebsiella pneumoniae (2) followed by an epidemic related to theK.pneumoniae carbapenemase (KPC) (1).Atpresent, theOXA-48carbapenemases comprise thefastestgrowinggroupofcarbapenemasesinEurope(3). InGreeceandItalyaround62and33%,respectively, of invasive K.pneumoniae are now non-susceptible to carbapenems (4). In 2015,13/38 countries reported inter-regional spread of or an endemic situation for carbapenemase-producing Enterobacteriaceae (CPE), comparedwith 6/38 in 2013. Only three countries repliedthat they had not identified one single case of CPE (3). Other particularly problematiccarbapenemasesaretheNewDelhimetallo-β-lactamases(NDMs),whicharehighlyprevalentontheIndiansubcontinentandintheMiddleEastandhaveonseveraloccasionsbeenimportedtoEurope (3), and in therearealsoexamplesof regionaldissemination in somecountries (5). TheIMP-carbapenemasesarealsocommoninsomepartsoftheworld(6).Carbapenemases are a source of concern because theymay confer resistance to virtually all β-lactams, and are readily transferable. Further, carbapenemase-producing strains frequentlypossess resistance mechanisms to a wide-range of antimicrobial agents, and infections withcarbapenemase-producingEnterobacteriaceaeareassociatedwithhighmortalityrates(7-9).2.3MechanismsofresistanceThevastmajorityof carbapenemasesareacquiredenzymes,encodedbygeneson transposableelements located on plasmids. Carbapenemases are expressed at various levels and differsignificantlyinbothbiochemicalcharacteristicsandactivityagainstspecificβ-lactams.Thelevelofexpressionandpropertiesoftheβ-lactamaseandthefrequentassociationwithotherresistancemechanisms (otherβ-lactamases,effluxand/oralteredpermeability) result in thewiderangeofresistance phenotypes observed among carbapenemase-producing isolates (10,11). Decreasedsusceptibility to carbapenems in Enterobacteriaceae may, however, also be caused by eitherextendedspectrumβ-lactamases(ESBL)orAmpCenzymescombinedwithdecreasedpermeabilitydue toalterationordown-regulationofporins (12), andpossiblyalsopenicillin-bindingproteins(13).

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Carbapenemase-producing Enterobacteriaceae (CPE) usually have decreased susceptibility tocarbapenems, and inmost cases are resistant to extended-spectrum (oxyimino) cephalosporins(i.e. cefotaxime, ceftriaxone, ceftazidime and/or cefepime) (14). However, with some enzymes(e.g. OXA-48-like) the organisms may be fully susceptible to cephalosporins. Currently, themajorityof theCPE isolatesalsoco-expresscephalosporin-hydrolyzingenzymes, suchasCTX-M-typeESBLs,andarethenalsoresistanttocephalosporins.Carbapenemasesareconsideredtobeof high epidemiological importance, particularly when they confer decreased susceptibility tocarbapenems(imipenem,meropenem,ertapenemanddoripenem),i.e.whentheMICsareabovetheepidemiologicalcut-off(ECOFF)valuesdefinedbyEUCAST(15).2.4RecommendedmethodsfordetectionofcarbapenemasesinEnterobacteriaceae2.4.1Screeningforcarbapenemase-productionCarbapenemMICs for carbapenemase-producing Enterobacteriaceaemay be below the clinicalbreakpoints (14-16). However, the ECOFF values as defined by EUCAST can be used to detectcarbapenemase-producers. Meropenem offers the best compromise between sensitivity andspecificity in terms of detecting carbapenemase-producers (14, 17). Ertapenem is the mostsensitivecarbapenem,buthaslowspecificity,especiallyinspeciessuchasEnterobacterspp.,dueto its relatively scarce instability to extended-spectrum β-lactamases (ESBLs) and AmpCβ-lactamasesincombinationwithporinloss(14).Appropriatecut-offvaluesfordetectingputativecarbapenemase-producers are shown in Table 1. It should be noted that in order to increasespecificity, imipenem and ertapenem screening cut-off values are one-dilution step higher thanthecurrentlydefinedECOFFs.Table 1. Clinical breakpoints and screening cut-off values for carbapenemase-producingEnterobacteriaceae(accordingtoEUCASTmethodology).

Carbapenem MIC(mg/L) Diskdiffusionzonediameter

(mm)with10µgdisksS/Ibreakpoint Screening

cut-offS/Ibreakpoint Screeningcut-

offMeropenem1 ≤2 >0.125 ≥22 <282

Ertapenem3 ≤0.5 >0.125 ≥25 <25

1Bestbalanceofsensitivityandspecificity2Isolateswith25-27mmonlyneedtobeinvestigatedforcarbapenemase-productioniftheyareresistanttopiperacillin-tazobactamand/ortemocillin(temocillincontributesmoretothespecificity).Investigationforcarbapenemasesisalwayswarrantedifzonediameterofmeropenemis<25mm.

3Highsensitivitybut lowspecificity.Canbeusedasanalternativescreeningagent,but isolateswithESBLandAmpCmayberesistantwithouthavingcarbapenemases.

Following detection of reduced susceptibility to carbapenems in routine susceptibility tests,phenotypicmethodsfordetectionofcarbapenemasesshouldbeapplied.Themaincategoriesofmethods are the combination disk test methods, colorimetric assays based on hydrolysis of

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carbapenems,othermethodsdetectingcarbapenemhydrolysis,andfinallylateralflowassays.Thevarioustestsaredescribedbelow.2.4.2CombinationdisktestingThecombinationdisktestmethodiscommerciallyavailablefromseveralmanufacturers,andwasthe firstphenotypic test tobecomeavailable (MAST,UK;Rosco,Denmark) (18-20).Thedisksortablets contain meropenem ± various inhibitors. In brief, boronic acid inhibits class Acarbapenemases (although data beyond KPC are scarce), and dipicolinic acid andethylenediaminetetraacetic acid (EDTA) inhibit class B carbapenemases. Moreover, OXA-48 isinhibitedbyavibactam,whichhasneverthelesssofarnotbeenincludedinthephenotypicpanels(21, 22). Cloxacillin, which inhibits AmpC β-lactamases, has been added to the tests todifferentiatebetweenAmpChyperproductionplusporinlossandcarbapenemase-production.Thealgorithmfor interpretationoftheseinhibitortests isoutlinedinFigure1andTable2.Themaindisadvantagewiththesemethodsisthattheytake18hours(inpracticeovernightincubation),forwhichreasonnovelrapidmethodshavebeenintroduced.Figure1.Algorithmforcarbapenemasedetection.

1Combination of several carbapenemases can also contribute to no synergy – e.g. MBL and KPC incombination.Moleculartestingisusuallynecessaryinsuchcases

2High-leveltemocillinresistance(>128mg/L,zonediameter<11mm)isaphenotypicmarkerofOXA-48The algorithm in Table 2 differentiates betweenmetallo-β-lactamases, classA carbapenemases,classDcarbapenemasesandnon-carbapenemases(ESBLand/orAmpCplusporinloss).Thetestscan be donewith the EUCAST disk diffusionmethod for non-fastidious organisms. Commercialtestsshouldbesetupaccordingtothemanufacturer’sinstructionsforeachtest.At present there are no available inhibitors for OXA-48-like enzymes. Temocillin high-levelresistance(MIC>128mg/L)hasbeenproposedasaphenotypicmarker forputativeOXA-48-likecarbapenemase producers (23, 24). However, this marker is not specific for OXA-48-typecarbapenemases asother resistancemechanismsmight confer this phenotype. ThepresenceofOXA-48-likeenzymesthereforehastobeconfirmedwithothermethods.Useofthemodifiedcloverleaf(Hodge)testisnotcurrentlyrecommendedasresultsaredifficulttointerpret, thespecificity ispoor,and in somecases thesensitivity isalsosuboptimal (12).Some

Synergy with boronic acid only

Synergy with boronic acid and cloxacillin

Synergy dipicolinic acid only

KPC (or other class A carbapenemase)

AmpC (chromosomal or plasmid-mediated)

plus porin loss

Metallo-β-lactamase (MBL)

No synergy1

Temocillin R2: OXA-48

Exception: meropenem 25-27 mm AND piperacillin-tazobactam=I/S:

no further testing

Temocillin S: ESBL plus porin

loss

Meropenem <28 mm with disk diffusion (or MIC >0.125 mg/L)

in all Enterobacteriaceae

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novelmodificationsof the techniquehavebeendescribed,but theyarecumbersome foruse inroutineclinicallaboratoriesanddonotsolveallproblemsofsensitivityandspecificity.Table2.Interpretationofphenotypictests(carbapenemasesinboldtype)bydiffusionmethodswithdisksortablets.Theexactdefinitionsofsynergyareprovidedinpackageinsertsforthevariouscommercialproducts.

Β-lactamase

Synergyobservedasincreaseinzonediameter(mm)with10µgmeropenemdisk/tablet

TemocillinMIC>128mg/Lorzonediameter<11mm

DPA/EDTA APBA/PBA DPA+APBA CLX

MBL + - - - Variable1

KPC - + - - Variable1

MBL+KPC2 Variable Variable + - Variable1

OXA-48-like - - - - Yes

AmpC+porinloss - + - + Variable1

ESBL+porinloss - - - - NoAbbreviations:MBL=metallo-β-lactamase,KPC=Klebsiellapneumoniaecarbapenemase,DPA=dipicolinicacid,EDTA=ethylenediaminetetraaceticacid,APBA=aminophenylboronicacid,PBA=phenylboronicacid,CLX=cloxacillin.1Temocillinsusceptibilitytestisrecommendedonlyincaseswherenosynergyisdetected,inordertodifferentiatebetweenESBL+porinlossandOXA-48-likeenzymes(23,24).Whenotherenzymesarepresentthesusceptibilityisvariableanddoesnotprovideanyfurtherindicationoftheβ-lactamasepresent.

2Thereisonereportsupportingtheuseofcommercialtabletscontainingdoubleinhibitors(DPAorEDTAplusAPBAorPBA)(25),butmulti-centrestudiesormultiplesinglecentrestudiesarelacking.Thiscombinationconfershigh-levelresistancetocarbapenemsandisrareoutsideGreece.

2.4.3Biochemical(colorimetric)testsTheCarbaNPtestisarapid(<2h)testfordetectionofcarbapenemhydrolysis,whichwillgiverisetoapH-changeresultinginacolourshiftfromredtoyellowwithphenolredsolution(26,27).TheCarbaNP test has been validatedwith bacterial colonies grown onMueller-Hinton agar plates,blood agar plates, trypticase soy agar plates, and most selective media used in screening forcarbapenemase producers. The Carba NP test should not be performedwith bacterial coloniesgrownonDrigalskiorMcConkeyagarplates.Thedifferent steps in themethodmustbecloselyfollowedinordertoobtainreproducibleresults.Severalpublicationsindicateahighsensitivityandspecificity of the method (28), whereas one publication observed problems of sensitivity forisolateswithamucoidphenotypeandforsomeEnterobacteriaceaeproducingOXA-48(29).Onecommercial variant of the method has been shown to perform well for detection ofcarbapenemases in Enterobacteriacae (30, 31). With certain commercial assays, including thecommercial version of the CarbaNP test, there are some interpretation issues based on visualreading of the colour shift with doubtful results and a certain proportion (3-5%) of non-interpretableresults.

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AderivativeoftheCarbaNP-test,theBlue-Carbatest(BCT)isabiochemicaltestforrapid(<2h)detectionofcarbapenemaseproduction(32,33).Itisbasedontheinvitrohydrolysisofimipenembybacterialcolonies(directinoculationwithoutpriorlysis),whichisdetectedbychangesinpHvaluesrevealedbytheindicatorbromothymolblue(bluetogreen/yelloworgreentoyellow).InalargeevaluationcarriedoutbyPasteranetal.(34),butconductedinonlyasinglelaboratory,thetestwasfoundtohaveexcellentsensitivityforclassAandBenzymes,butsuboptimalsensitivityfordetectionofOXA-48enzymes. A thirdbiochemical test is theβCARBA test™,whichalsocanbecarriedout in<2h.The test isconductedbymixing1to3coloniesinthereagents.Readingsshouldbedoneafteramaximumof30minof incubation.Changeofcolor fromyellowtoorange, redorpurple indicatedapositivereaction.Onestudyfoundthatthe0.5-hourincubationtimerecommendedbythemanufactureristoo short forOXA-48-producing strains. Theevaluated strain collectionwasquite limited,andalargertrialwassuggestedtoinvestigatehowthetestcomparestootherbiochemicaltests(35).Inanotherevaluation,theβCarbatest™showedexcellentperformancetodetectCPE,andespeciallyOXA-48.However, theability todetectotherclassAcarbapenemasesshouldbe furtherverifiedand some false-positive results occurredwith other β-lactamases such as overproduction of K1β-lactamaseinKlebsiellaoxytoca(33).2.4.4 CarbapenemInactivationMethodTheprincipleofthismethodistodetectenzymatichydrolysisbyincubatingacarbapenemwithabacterial suspension. The CIM-test utilizes antibiotic susceptibility-testing disks as substratealiquots.Followingtwohoursofincubationofafullloopofbacteriawithameropenemdisk,thediskisplacedonanagarinoculatedwithEscherichiacoliATCC25922.Enzymaticinactivationwillproduce no zone, whereas no carbapenemase activity will imply there will be a zone, asmeropenem in thediskhasnotbeenhydrolyzed.TheCIM-testhashadvariableperformance indifferent studies (36-38), but remains a possible alternative, although the negative predictivevalue of the test is still not clear. One main disadvantage of this technique is that it requiresusuallyatleast18hourstoobtaintheresults.2.4.5 DetectionofcarbapenemhydrolysiswithMALDI-TOFThe principle is to detect in a mass spectrometry device (MALDI TOF) the decrease ordisappearance of certain specific peaks of carbapenems in a mass spectra when a bacterialsuspension is previously incubated with the carbapenem (39, 40). Spectra are measured afterdryingbetweenm/z160and600,usingaMicroflexLTmassspectrometer(39).Themethodhasbeenfoundinseveralstudiestohavegoodsensitivityandspecificity,butexceptforOXA-48-groupenzymes.Toamendthisproblem,NH4HCO3canbeaddedto thereaction,andthismodificationwasshowninonestudytoimprovedetectionofOXA-48(41).However,thenewmethodhasthusfarnotbeenevaluatedinamulticenterstudydesignorinmultiplesinglecenterstudies.AnotherslightlyimpracticalfeatureisthatthesettingsfortheMALDI-TOFneedtobealteredascomparedtowhatisusedforspeciesdetermination(41). LateralflowassaysAnew immunochromatographic lateral flowassaywas recently described. The test is basedonimmunological capture of epitopes of OXA-48, using colloidal gold nanoparticles bound to anitrocellulosemembranewithinalateralflowdevice.Theprincipleofthetestisthatmonoclonalanti-OXA-48antibodiesareselectedasspecificcapturereagents,fordirectidentificationOXA-48-like enzymes (42). The assay takes around four minutes, and has been evaluated both fromcoloniesand fromspikedbloodculturebottles (43-46).Recently, a similar test forKPChasalso

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beendeveloped,but its robustnesshasnotbeenevaluatedbymultiple single center studiesoronemulticenterstudy(46).2.4.6 ControlstrainsBelowsomepossiblecontrol strains forphenotypicandgenotypicexperimentshavebeen listedfor convenience.Quality control rangesarenot available for these strains.Usersof commercialmethodsshouldstudythepackageinsertforinformationonwhichcontrolstrainstouse.Table3.Examplesofcontrolstrainsforcarbapenemasetesting.

Strain Mechanism

EnterobactercloacaeCCUG59627 AmpCcombinedwithdecreasedporinexpression

K.pneumoniaeCCUG58547orK.pneumoniaeNCTC13440 Metallo-β-lactamase(VIM)

K.pneumoniaeNCTC13443 Metallo-β-lactamase(NDM-1)

E.coliNCTC13476 Metallo-β-lactamase(IMP)K.pneumoniaeCCUG56233orK.pneumoniaeNCTC13438 Klebsiellapneumoniaecarbapenemase(KPC)

K.pneumoniaeNCTC13442 OXA-48carbapenemase

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37. YamadaK, KashiwaM,Arai K,NaganoN, SaitoR. Comparisonof theModified-Hodge test, CarbaNP test, andcarbapenem inactivation method as screening methods for carbapenemase-producing Enterobacteriaceae. JMicrobiolMethods.2016;128:48-51.

38. Tijet N, Patel SN, Melano RG. Detection of carbapenemase activity in Enterobacteriaceae: comparison of thecarbapeneminactivationmethodversustheCarbaNPtestJAntimicrobChemother.2016;71(1):274-6.

39. HrabákJ,StudentováV,WalkováR,ZemlickováH,JakubuVetal.DetectionofNDM-1,VIM-1,KPC,OXA-48,andOXA-162carbapenemasesbymatrix-assisted laserdesorption ionization-timeofflightmassspectrometry.JClinMicrobiol.2012;50:2441-3

40. LasserreC,DeSaintMartinL,CuzonG,BogaertsP,LamarE,etal.EfficientDetectionofCarbapenemaseActivityinEnterobacteriaceaebyMatrix-AssistedLaserDesorptionIonization-TimeofFlightMassSpectrometryinLessThan30Minutes.JClinMicrobiol.2015;53:2163-71.

41. Papagiannitsis CC, Študentová V, Izdebski R, Oikonomou O, Pfeifer Y, et al. Matrix-assisted laser desorptionionization-timeofflightmassspectrometrymeropenemhydrolysisassaywithNH4HCO3,areliabletoolfordirectdetectionofcarbapenemaseactivity.JClinMicrobiol.2015;53(5):1731

42. PasteranF,DenormeL,OteI,GomezS,DeBelderD,GlupczynskiY,BogaertsP,GhiglioneB,PowerP,MertensP,CorsoA.Rapid identificationofOXA-48andOXA-163Subfamilies incarbapenem-resistantgram-negativebacilliwithanovelimmunochromatographiclateralflowassay.JClinMicrobiol.2016;54(11):2832-2836

43. Dortet L, Jousset A, Sainte-Rose V, Cuzon G, Naas T. Prospective evaluation of the OXA-48 K-SeT assay, animmunochromatographictestfortherapiddetectionofOXA-48-typecarbapenemases.JAntimicrobChemother.2016;71(7):1834-40.

44. WarehamDW,ShahR,Betts JW,PheeLM,MominMH.Evaluationofan ImmunochromatographicLateralFlowAssay(OXA-48K-SeT)forRapidDetectionofOXA-48-LikeCarbapenemasesinEnterobacteriaceae.JClinMicrobiol.2016;54(2):471-3

45. Meunier D, Vickers A, Pike R, Hill RL, Woodford N, Hopkins KL. Evaluation of the K-SeT R.E.S.I.S.T.immunochromatographic assay for the rapid detection of KPC and OXA-48-like carbapenemases. J AntimicrobChemother.2016;71(8):2357-9.

46. Glupczynski Y, Evrard S, Ote I, Mertens P, Huang TD, et al. Evaluation of two new commercialimmunochromatographic assays for the rapid detection of OXA-48 and KPC carbapenemases from culturedbacteria.JAntimicrobChemother.2016;71(5):1217-22

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3.Extended-spectrumβ-lactamase(ESBL)-producingEnterobacteriaceae

ImportanceofdetectionofresistancemechanismRequiredforclinicalantimicrobialsusceptibilitycategorization NoInfectioncontrolpurposes YesPublichealthpurposes Yes

3.1DefinitionESBLs are enzymes that hydrolyze most penicillins and cephalosporins, including oxyimino-β-lactamcompounds(cefuroxime,third-andfourth-generationcephalosporinsandaztreonam)butneithercephamycinsnorcarbapenems.MostESBLsbelongtotheAmblerclassAofβ-lactamasesand are inhibited by β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) and bydiazabicyclooctanones(avibactam)(1).3.2Clinicaland/orepidemiologicalimportanceThe first ESBL-producing strains were identified in 1983, and since then have been observedworldwide. This distribution is a result of the clonal expansion of producer organisms, thehorizontal transferofESBLgenesonplasmidsand, lesscommonly, theiremergencedenovo.Byfar themost clinically important groups of ESBLs are CTX-Menzymes, emerging since the early2000s,followedbySHV-andTEM-derivedESBLs(2-5).ESBLproductionhasbeenobservedmostlyinEnterobacteriaceae,first inhospitalenvironments,later in nursinghomes, and since around2000 in the community (outpatients, healthy carriers,sick and healthy animals, food products). The most frequently encountered ESBL-producingspecies are E.coli and K.pneumoniae. However, all other clinically relevant EnterobacteriaceaespeciesarealsocommonESBL-producers.TheprevalenceofESBL-positive isolatesdependsonarangeoffactorsincludingspecies,geographiclocality,hospital/ward,groupofpatientsandtypeofinfection,andlargevariationshavebeenreportedindifferentstudies(2,3,6,7).TheEARS-Netdatafor 2015 showed that the rate of invasiveK.pneumoniae isolates non-susceptible to the third-generation cephalosporins exceeded 25% or even 50% inmany European countries. Except forGreece and Italy with high proportion of KPC-type carbapenemase-producing isolates, most oftheseisolateswerepresumedtobeESBL-producersbasedonlocalESBLtestresults(8).3.3MechanismsofresistanceThe vastmajority of ESBLs are acquiredenzymes, encodedby genesonplasmids. The acquiredESBLsareexpressedatvariouslevels,anddiffersignificantlyinbiochemicalcharacteristicssuchasactivity against specific β-lactams (e.g. cefotaxime, ceftazidime, aztreonam). The level ofexpression and properties of an enzyme, and the co-presence of other resistancemechanisms(other β-lactamases, active efflux, altered permeability) result in the large variety of resistancephenotypesobservedamongESBL-positiveisolates(1-4,9-11).

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3.4RecommendedmethodsfordetectionofESBLsinEnterobacteriaceaeInmanyareas,ESBLdetectionandcharacterization is recommendedormandatory for infectioncontrolpurposes.TherecommendedstrategyforthedetectionofESBLsinEnterobacteriaceaeisbasedonnon-susceptibilitytoindicatoroxyimino-cephalosporins,followedbyphenotypic(andinsomecasesgenotypic)confirmationtests(Table1,Figure1).Ascreeningbreakpointof>1mg/L isrecommendedforcefotaxime,ceftriaxone,ceftazidime,andcefpodoxime,inaccordancewiththeguidelinesissuedbyEUCASTandCLSI(Table1)(12,13).TheEUCASTclinicalbreakpointforEnterobacteriaceaeisalsoSusceptible≤1mg/L(12).CefpodoximeisthemostsensitiveindividualindicatorcephalosporinfordetectionofESBLproductionandmaybeusedforscreening.However,itislessspecificthanthecombinationofcefotaxime(orceftriaxone)and ceftazidime (14, 15) and only the latter compounds are used in the confirmation testing.CorrespondingzonediametersfortheindicatorcephalosporinsareshowninTable1.Table1.ESBLscreeningmethodsforEnterobacteriaceae(13-19).

Method Antibiotic ConductESBL-testingif

Brothoragardilution1

Cefotaxime/ceftriaxoneANDCeftazidime

MIC>1mg/Lforeitheragent

Cefpodoxime MIC>1mg/L

Diskdiffusion1

Cefotaxime(5μg)or Inhibitionzone<21mm

Ceftriaxone(30μg) Inhibitionzone<23mm

ANDCeftazidime(10μg) Inhibitionzone<22mm

Cefpdoxime(10µg) Inhibitionzone<21mm

1Withallmethodstesteither(i)cefotaximeorceftriaxoneANDceftazidimeOR(ii)cefpodoximealone.

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Figure1.AlgorithmforphenotypicdetectionofESBLs

1 IfcefoxitinhasbeentestedandhasanMIC>8mg/L,performcefepime+/-clavulanicacidconfirmationtest2 Cannotbedeterminedaseitherpositiveornegative(e.g.ifagradientdiffusionstripcannotbereadduetogrowthbeyondtheMICrangeofthestriporthereisnoclearsynergyincombination-diskanddouble-disksynergytests).Inconfirmationwithcefepime+/-clavulanicacidisstillindeterminate,genotypictestingisrequired.

3.4.1ESBL-screeninginEnterobacteriaceaeA. Screening in group 1 Enterobacteriaceae (E.coli, Klebsiella spp., Raoultella spp., Pmirabilis,Salmonellaspp.,Shigellaspp.)TherecommendedmethodsforESBLscreeningingroup1Enterobacteriaceaearebrothdilution,agar dilution, disk diffusion or an automated system (13, 20, 21). It is required that bothcefotaxime(orceftriaxone)andceftazidimeareusedasindicatorcephalosporins,astheremaybelarge differences in MICs of cefotaxime (or ceftriaxone) and ceftazidime for different ESBL-producingisolates(14,22,23).The algorithm for screening and phenotypic ESBL confirmation methods for group 1EnterobacteriaceaethatarepositiveinscreeningtestsaredescribedinFigure1andTable2.B.Screening ingroup2Enterobacteriaceae(Enterobacterspp,Serratiaspp.,Citrobacterfreundii,Morganellamorganii,Providenciaspp,Hafniaalvei)Forgroup2EnterobacteriaceaeitisrecommendedthatESBLscreeningisperformedaccordingtothe methods described above for group 1 Enterobacteriaceae (Figure 1 and Table 3) (19).However, a very commonmechanism of cephalosporin resistance is derepressed chromosomalAmpCβ-lactamaseinthesespecies.SincecefepimeisstabletoAmpChydrolysis,itcanbeusedinphenotypictestingwithclavulanicacid.3.4.2PhenotypicconfirmationmethodsFour of the several phenotypic methods based on the in vitro inhibition of ESBL activity byclavulanic acid are recommended for ESBL confirmation: the combination disk test (CDT), thedouble-disksynergytest(DDST),theESBLgradienttest,andthebrothmicrodilutiontest(Tables2and3)(20,21,24). Inonemulti-centrestudy,theCDTshowedabetterspecificitythantheESBLgradient test but with comparable sensitivity (25). Manufacturers of automated susceptibility

ESBL SCREENING:I/R to one or both of cefotaxime and ceftazidime

(or cefpodoxime R)

ESBL CONFIRMATION1

with ceftazidime and cefotaxime+/- clavulanic acid

Group 1:E.coli, Klebsiella spp., P. mirabilis, Salmonella spp., Shigella spp.

Group 2: Enterobacteriaceae with inducible chromosomal AmpC: Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia stuartii, Serratia spp., Hafnia alvei.

ESBL CONFIRMATIONwith cefepime +/- clavulanic acid

Species dependent ESBL confirmation

Negative: No ESBL Indeterminate Positive: ESBL Negative: no ESBL Positive: ESBL

Yes

No No ESBL

Indeterminate2

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testing systems have implemented detection tests based on the inhibition of ESBL enzymes byclavulanic acid. Performanceof confirmationmethodsdiffers indifferent studies, dependingonthecollectionofstrainstestedandthedeviceused(17-19).A.Combinationdisktest(CDT)For each test, disks or tablets containing the cephalosporin alone (cefotaxime, ceftazidime,cefepime) and in combinationwith clavulanic acid are applied. The inhibition zone around thecephalosporindiskortabletcombinedwithclavulanicacidiscomparedwiththezonearoundthediskortabletwiththecephalosporinalone.Thetest ispositiveiftheinhibitionzonediameteris≥5mmlargerwithclavulanicacidthanwithout(Table3)(26,27).B.Double-disksynergytest(DDST)Diskscontainingcephalosporins(cefotaxime,ceftazidime,cefepime)areappliedtoplatesnexttoadisk with clavulanic acid (amoxicillin-clavulanic acid). A positive result is indicated when theinhibitionzonesaroundanyofthecephalosporindisksareaugmentedorthereisa‘keyhole’inthedirection of the disk containing clavulanic acid. The distance between the disks is critical and20mmcentre-to-centrehasbeen foundtobeoptimal forcephalosporin30µgdisks;however itmay be reduced (15 mm) or expanded (30 mm) for strains with very high or low levels ofresistance,respectively(20).Therecommendationsneedtobere-evaluatedfordiskswithlowercephalosporincontent,asusedintheEUCASTdiskdiffusionmethod.C.GradienttestmethodESNL-specific gradient tests are set up, read and interpreted according to the manufacturer’sinstructions.Thetestispositiveif≥8-foldreductionisobservedintheMICofthecephalosporincombinedwithclavulanicacidcomparedwiththeMICofthecephalosporinaloneorifaphantomzone or deformed ellipse is present (see instructions from the manufacturer for illustrations)(Table3).Thetest result is indeterminate if thestripcannotbereadduetogrowthbeyondtheMICrangeofthestrip.Inallothercasesthetestresultisnegative.Accordingtothemanufacturer,ESBLgradienttestshouldbeusedforconfirmationofESBLproductiononlyandisnotreliablefordeterminationoftheMIC.D.BrothmicrodilutionBrothmicrodilutionisperformedwithcation-adjustedMueller-Hintonbrothcontainingserialtwo-folddilutionsofcefotaxime,ceftazidimeandcefepimeatconcentrationsrangingfrom0.25to512mg/L,withandwithoutclavulanicacidata fixedconcentrationof4mg/L.Thetest ispositive if≥8-foldreduction isobserved intheMICofanyofthecephalosporinscombinedwithclavulanicacidcomparedwiththeMICofthatcephalosporinalone,otherwisethetestresultisinterpretedasnegative(24).E.Biochemical(colorimetric)testingTheESBLNDPtestwasdescribedfirstin2012,andusescefotaximeasindicatorantimicrobial,withtazobactamasinhibitor(28).Itiscarriedoutin96-wellplatesorinseparatetubes.Colourchangefrom red to yellow is regarded a positive test. The test has also been used directly on patientsamples (29). Excellent sensitivities and specificities have been described, but the test has notbeenevaluatedinanymulticentertrial.Theβ-LACTAtestisacolorimetrictestusingachromogeniccephalosporinsubstrate(HMRZ-86)onisolatesandalsodirectlyonclinical samples (30). Inaprospectivemulticentric study inBelgiumand France, it was found to display an excellent sensitivity and specificity for E.coli and

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K.pneumoniae(96%and100%,respectively)whileitshowedalowersensitivity(67%)forspeciesproducing inducible AmpC β-lactamases. The high negative predictive value for E.coli andK.pneumoniae(99%inareaswithprevalenceofC3Gresistancerangingbetween10-30%)makesthissimpletestveryefficientforthepredictionofresistancetothird-generationcephalosporins,particularlyinextended-spectrumβ-lactamase-producingstrains.E.SpecialconsiderationsininterpretationESBLconfirmationteststhatusecefotaximeastheindicatorcephalosporinmaybefalse-positiveforKlebsiellaoxytocastrainswithhyperproductionofthechromosomalK1(OXY-like)β-lactamases(31). A similar phenotype may also be encountered in Proteus vulgaris, Proteus penneri,Citrobacter koseri and Kluyvera spp. and in some C. koseri-related species like C.sedlakii,C.farmeri and C.amalonaticus, which have chromosomal β-lactamases that are inhibited byclavulanic acid (32, 33). Another possible cause of false-positive results is hyperproduction ofSHV-1-, TEM-1- orOXA-1-like broad-spectrumβ-lactamases combinedwith alteredpermeability(18). Similar problems with false-positive test results for K1-producing K.oxytoca or for OXA-1producingE.colimayalsoarisewhenusingconfirmationtestsbasedoncefepimeonly(34).Table2.ESBLconfirmationmethodsforEnterobacteriaceaethatarepositiveintheESBLscreeningtest(seeTable1).Group1Enterobacteriaceae(seeFigure1).

Method Antimicrobialagent(diskcontent) ESBLconfirmationispositiveif

ESBLgradienttest Cefotaxime+/-clavulanicacid

MICratio≥8ordeformedellipsepresent

Ceftazidime+/-clavulanicacid

MICratio≥8ordeformedellipsepresent

Combinationdiskdiffusiontest(CDT)

Cefotaxime(30µg)+/-clavulanicacid(10µg)

≥5mmincreaseininhibitionzone

Ceftazidime(30µg)+/-clavulanicacid(10µg)


Brothmicrodilution

Cefotaxime+/-clavulanicacid(4mg/L)

MICratio≥8

Ceftazidime+/-clavulanicacid(4mg/L)

MICratio≥8

Cefepime+/-clavulanicacid(4mg/L)

MICratio≥8

Doubledisksynergytest(DDST)

Cefotaxime,ceftazidimeandcefepime

Expansionofindicatorcephalosporininhibitionzonetowardsamoxicillin-clavulanicaciddisk

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Table3.ESBLconfirmationmethodsforEnterobacteriaceaethatarepositiveintheESBLscreening(seeTable1).Group2Enterobacteriaceae(seeFigure1).

Method Antibiotic ConfirmationispositiveifESBLgradienttestEtest®®ESBL

Cefepime+/-clavulanicacid MICratio≥8ordeformedellipsepresent

Combinationdiskdiffusiontest

Cefepime(30µg)+/-clavulanicacid(10µg)


Brothmicrodilution

Cefepime+/-clavulanicacid(fixedconcentration4mg/L)

MICratio≥8

Doubledisksynergytest(DDST)

Cefotaxime,ceftazidime,Cefepime

Expansionofindicatorcephalosporininhibitionzonetowardsamoxicillin-clavulanicaciddisk

3.4.3PhenotypicdetectionofESBLinthepresenceofotherβ-lactamasesthatmasksynergyIndeterminate test results (Etest®) and false-negative test results (CDT, DDST, Etest® and brothmicrodilution)mayresult fromthehigh-levelexpressionofAmpCβ-lactamases,whichmaskthepresenceofESBLs (20,34,35). Isolateswithhigh-levelexpressionofAmpCβ-lactamasesusuallyshowclearresistancetothird-generationcephalosporins.Inaddition,resistancetocephamycins,e.g.acefoxitinMIC>8mg/L,maybeindicativeofhigh-levelexpressionofAmpCβ-lactamases(34),withtherareexceptionofACCβ-lactamases,whichdonotconfercefoxitinresistance(36).ToconfirmthepresenceofESBLsinisolateswithhigh-levelexpressionofAmpCβ-lactamasesitisrecommended that an additional ESBL confirmation test be performed with cefepime as theindicatorcephalosporin,ascefepimeisgenerallynothydrolyzedbyAmpCβ-lactamases.Cefepimemay be used in all the CDT, DDST, gradient test or broth dilution test formats (27, 37-39).Alternativeapproachesincludeuseofcloxacillin,whichisagoodinhibitorofAmpCenzymes.Testformats include CDT with disks containing the two cephalosporin indicators (cefotaxime andceftazidime)withbothclavulanicacidandcloxacillintogether,andstandardCDTorDDSTonagarplatessupplementedwith200-250mg/Lcloxacillin(19).Therearealsodisksortabletscontainingbothclavulanicacidandcloxacillinonthemarket,butthereisinsufficientpublishedvalidationoftheseproducts.Thepresenceof ESBLsmayalsobemaskedby carbapenemases suchasMBLsorKPCs (butnotOXA-48-likeenzymes)and/orseverepermeabilitydefects(40,41). Ifdetection isstillconsideredrelevantinsuchcases,molecularmethodsshouldbeusedforESBLdetection.3.4.4GenotypicconfirmationForthegenotypicconfirmationofthepresenceofESBLgenesthereareanumberofpossibilitiesavailable ranging from PCR and sequencing to whole-genome sequencing, followed by in silicomappingofresistancegenes.Therearealsodifferentmicroarraysavailable.Bothcommercialandin-housemethodsexist,buttheyhavenotbeensystematicallyexaminedandwillthereforenotbeelaborateduponinthisdocument.Whole-genomesequencingapproacheshavebeendescribedinanotherEUCASTpublication(42).

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3.4.5QualitycontrolBelowsomepossiblecontrol strains forphenotypicandgenotypicexperimentshavebeen listedfor convenience.Quality control rangesarenot available for these strains.Usersof commercialmethodsshouldstudythepackageinsertforinformationonwhichcontrolstrainstouse.Table4.ExamplesofcontrolstrainsforESBLtesting.

Strain Mechanism

K.pneumoniaeATCC700603 SHV-18ESBL

E. coliCCUG62975 CTX-M-1groupESBLandacquiredCMYAmpC

E.coliATCC25922 ESBL-negative

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38. Yang JL, Wang JT, Lauderdale TL, Chang SC. Prevalence of extended-spectrum β-lactamases in Enterobactercloacae in Taiwan and comparison of 3 phenotypic confirmatorymethods for detecting extended-spectrumβ-lactamaseproduction.JMicrobiolImmunolInfect.2009;42:310-6.

39. Jeong SH, Song W, Kim JS, Kim HS, Lee KM. Broth microdilution method to detect extended-spectrum β-lactamasesandAmpCβ-lactamases inenterobacteriaceae isolatesbyuseofclavulanicacidandboronicacidasinhibitors.JClinMicrobiol.2009;47:3409-12.

40. Tsakris A, PoulouA, Themeli-Digalaki K, Voulgari E, Pittaras T, SofianouD, Pournaras S, PetropoulouD.Use ofboronic acid disk tests to detect extended- spectrum β-lactamases in clinical isolates of KPC carbapenemase-possessingEnterobacteriaceae.JClinMicrobiol.2009;47:3420-6.

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41. MarchA,AschbacherR,DhanjiH,LivermoreDM,BöttcherAetal.Colonizationofresidentsandstaffofa long-term-carefacilityandadjacentacute-carehospitalgeriatricunitbymultiresistantbacteria.ClinMicrobiolInfect.2010;16:934-44.

42. EllingtonMJ,EkelundO,AarestrupFM,etal.Theroleofwholegenomesequencinginantimicrobialsusceptibilitytestingofbacteria:reportfromtheEUCASTSubcommittee.ClinMicrobiolInfect.2017;23(1):2-22.

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4.AcquiredAmpCβ-lactamase-producingEnterobacteriaceae


4.1DefinitionAmpC-type cephalosporinases are Ambler class C β-lactamases. They hydrolyze penicillins,cephalosporins (including the third-generation but generally not the fourth-generationcompounds) and monobactams. In general, AmpC-type enzymes are poorly inhibited by theclassicalESBLinhibitors,especiallyclavulanicacid(1).4.2Clinicaland/orepidemiologicalimportanceThefirstisolatesproducingacquiredAmpCswereidentifiedduringthe1980s,andsincethentheyhavebeenobservedgloballyasa resultof clonal spreadandhorizontal transferofAmpCgenes(oftenreferredtoasplasmid-mediatedAmpC).ThereareseverallineagesofmobileAmpCgenes,originating from natural producers, namely the Enterobacter group (MIR, ACT), the C.freundiigroup (CMY-2-like, LAT, CFE), theM.morganii group (DHA), the Hafnia alvei group (ACC), theAeromonas group (CMY-1-like, FOX, MOX) and the Acinetobacter baumannii group (ADC). ThemostprevalentandmostwidelydisseminatedaretheCMY-2-likeenzymes,althoughtheinducibleDHA-likeβ-lactamasesandsomeothershavealsospreadextensively(1).ThemajorproducerspeciesofacquiredAmpCsareE.coli,K.pneumoniae,SalmonellaentericaandP.mirabilis. Isolates with these enzymes have been recovered from both hospitalized andcommunitypatients,andtheywererecognizedearlierthanclassicalESBL-enzymesinfarmanimalsand infoodproducts(inE.coliandS.enterica).AlthoughtheacquiredAmpCshavebeenspreadwidelyandbeenrecordedinmulti-centrestudiesofenterobacterialresistancetothird-generationcephalosporins,theiroverallfrequencyhasremainedfarbelowthatofESBLs,at least inEurope.However, in some local and specific epidemiological settings, the significance of organismsproducingtheseenzymesmaysubstantiallyincrease(1-5).4.3MechanismsofresistanceNumerous Enterobacteriaceae and some other Gram-negative bacilli produce natural AmpCs,eitherconstitutivelyatatracelevel(e.g.E.coli,Shigellaspp.)orinducibly(e.g.Enterobacterspp.,C.freundii,M.morganii,P.aeruginosa).ThederepressionorhyperproductionofnaturalAmpCsisdue to various genetic changes and confers high-level resistance to cephalosporins and topenicillin-β-lactamase inhibitor combinations. The class C cephalosporinases can also occur asacquired enzymes, mainly in Enterobacteriaceae. Except for the inducible DHA enzyme, theacquired AmpCs are expressed constitutively, conferring resistance similar to that in thederepressedorhyperproducingmutantsofnaturalAmpCproducers.Resistancelevelsdependonthe amounts of enzymes expressed, as well as the presence of other resistance mechanisms.SimilartoESBLs,theacquiredAmpCgenesareusuallyfoundonplasmids(1-3).

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4.4RecommendedmethodsfordetectionofacquiredAmpCinEnterobacteriaceaeAcefoxitinMIC>8mg/L(zonesize<19mm)combinedwithphenotypicresistancetoceftazidimeand/orcefotaxime(asdefinedbybreakpoints)maybeusedasphenotypiccriteriaforinvestigationofAmpCproductioningroup1Enterobacteriaceae,althoughthisstrategywillnotdetectACC-1,aplasmid-mediatedAmpC that doesnot hydrolyze cefoxitin (6). It shouldbenoted that cefoxitinresistancemayalsobeduetoporindeficiency(1).Figure1.AlgorithmforAmpCdetection.

1 Cefoxitin ‘R’ isheredefinedasnon-wild type (MIC>8mg/Lor zonediameter<19mm).Forcefotaximeandceftazidime‘R’istheresultobtainedusingthecurrentEUCASTbreakpoints.Investigationofisolateswith non-susceptibility to cefotaxime and ceftazidime is an approachwith higher sensitivity but lowerspecificitycomparedwithfocusingoncefoxitinresistantisolates(7).AmpCcanalsobepresentinisolateswithapositiveESBL-test(clavulanicacidsynergy).Forlaboratoriesnottestingcefoxitin,susceptibilitytocefepime togetherwith resistance to cefotaximeand/or ceftazidime is anotherphenotypic indicator ofAmpC,althoughlessspecific.

Phenotypic AmpC confirmation tests are generally based on inhibition of AmpC by eithercloxacillin or boronic acid derivatives. However, boronic acid derivatives also inhibit class Acarbapenemases, aswell as some class A penicillinases such as K1 inK.oxytoca. Although dataevaluating these methods is sparse, reasonably accurate detection with in-house methods hasbeen described (8-10) as well as with commercially available tests such as the Mast “AmpCDetectionDiscSet” (sensitivity96-100%,specificity98%-100%) (11,12), theAmpCgradienttest,currently available only from bioMérieux (sensitivity 84-93%, specificity 70-100%) (12, 13) andRosco tablets with cefotaxime-cloxacillin and ceftazidime-cloxacillin (sensitivity 96%, specificity92%)(7,14).ForE.coli,however,AmpCconfirmationtestscannotdiscriminatebetweenacquiredAmpCandconstitutivehyperproductionofthechromosomalAmpC.Thepresenceof acquiredAmpCs speciesmay alsobe confirmedusingPCR-basedmethods (15,16),orwithDNAmicroarray-basedmethod(17).

Cefotaxime R or ceftazidime R AND cefoxitin R1

in E. coli, K. pneumoniae, P. mirabilis, Salmonella spp,

Shigella spp

Cloxacillin synergydetected

Cloxacillin synergynot detected

E. coli and Shigella spp: PCR is required to discriminatebetween plasmid-acquiredand chromosomal AmpC

Other mechanisms(e.g. porin loss)

K.pneumoniae, P. mirabilis, Salmonella (lack chromo-

somal AmpC) plasmid-mediated AmpC detected

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Belowsomepossiblecontrol strains forphenotypicandgenotypicexperimentshavebeen listedfor convenience.Quality control rangesarenot available for these strains.Usersof commercialmethodsshouldrefertothepackageinsertforinformationonwhichcontrolstrainstouse.Table1.ExamplesofcontrolstrainsforAmpCtesting.

Strain Mechanism

E.coliCCUG58543 AcquiredCMY-2AmpC

E.coliCCUG62975 AcquiredCMYAmpCandCTX-M-1groupESBL

K.pneumoniaeCCUG58545 AcquiredDHA

E.coliATCC25922 AmpCandESBLnegative

4.5References1. JacobyGA.AmpCβ-lactamases.ClinMicrobiolRev.2009;22:161-822. Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type β-lactamases. Antimicrob Agents Chemother.

2002;46:1-113. BeceiroA,BouG.ClassCβ-lactamases:anincreasingproblemworldwide.RevMedMicrobiol.2004;15:141-1524. Empel J, Hrabák J, Kozińska A, Bergerová T, Urbášková P, Kern-Zdanowicz I, GniadkowskiM. DHA-1-producing

KlebsiellapneumoniaeinateachinghospitalintheCzechRepublic.MicrobDrugResist.2010;16:291-2955. D’AndreaMM,LiterackaE,ZiogaA,GianiT,BaraniakA,FiettJ,SadowyE,TassiosPT,RossoliniGM,Gniadkowski

M, Miriagou V. Evolution of a multi-drug-resistant Proteus mirabilis clone with chromosomal AmpC-typecephalosporinasesspreadinginEurope.AntimicrobAgentsChemother.2011;55:2735-2742

6. BauernfeindA,SchneiderI,JungwirthR,SahlyH,UllmannU.AnoveltypeofAmpCβ-lactamase,ACC-1,producedbyaKlebsiellapneumoniaestraincausingnosocomialpneumonia.AntimicrobAgentsChemother.1999;43:1924-31.

7. Edquist P, RingmanM, Liljequist BO, Wisell KT, Giske CG. Phenotypic detection of plasmid-acquired AmpC inEscherichiacoli-evaluationofscreeningcriteriaandperformanceoftwocommercialmethodsforthephenotypicconfirmationofAmpCproduction.EurJClinMicrobiolInfectDis.2013;32:1205-10

8. Yagi T,Wachino J, KurokawaH, Suzuki S, YamaneKet al. Practicalmethodsusingboronic acid compounds foridentification of class C β-lactamase-producing Klebsiella pneumoniae and Escherichia coli. J Clin Microbiol.2005;43:2551-8.

9. Tenover FC, Emery SL, Spiegel CA, Bradford PA, Eells S et al. Identification of plasmid-mediated AmpC β-lactamasesinEscherichiacoli,Klebsiellaspp.,andProteusspp.canpotentiallyimprovereportingofcephalosporinsusceptibilitytestingresults.JClinMicrobiol.2009;47:294-9.

10. Tan TY, Ng LS, He J, Koh TH, Hsu LY. Evaluation of screening methods to detect plasmid-mediated AmpC inEscherichiacoli,Klebsiellapneumoniae,andProteusmirabilis.AntimicrobAgentsChemother.2009;53:146-9

11. Martínez-Martínez L. Extended-spectrum β-lactamases and the permeability barrier. Clin Microbiol Infect.2008;14Suppl1:82-9.

12. Halstead FD, Vanstone GL, Balakrishnan I. An evaluation of the Mast D69C AmpC Detection Disc Set for thedetectionofinducibleandderepressedAmpCβ-lactamases.JAntimicrobChemother.2012;67:2303-4.

13. IngramPR, Inglis TJ, Vanzetti TR, Henderson BA,HarnettGB,Murray RJ. Comparison ofmethods for AmpC β-lactamasedetectioninEnterobacteriaceae.JMedMicrobiol.2011;60(Pt6):715-21.

14. Peter-GetzlaffS,PolsfussS,PoledicaM,HombachM,GigerJetal.DetectionofAmpCβ-lactamaseinEscherichiacoli:comparisonofthreephenotypicconfirmationassaysandgeneticanalysis.JClinMicrobiol.2011;49:2924-32.

15. HansenF,HammerumAM,SkovRL,GiskeCG,SundsfjordA,SamuelsenO.EvaluationofROSCONeo-Sensitabsforphenotypic detection and subgrouping of ESBL-, AmpC- and carbapenemase-producing Enterobacteriaceae.APMIS.2012;120:724-32.

16. Pérez-PérezFJ,HansonND.Detectionofplasmid-mediatedAmpCβ-lactamasegenesinclinical isolatesbyusingmultiplexPCR.JClinMicrobiol.2002;40:2153-62.

17. BrolundA,Wisell KT, Edquist PJ, Elfström L,WalderM,Giske CG.Development of a real-time SYBRGreen PCRassayforrapiddetectionofacquiredAmpCinEnterobacteriaceae.JMicrobiolMethods.2010;82:229-33.

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18. CuzonG,NaasT,BogaertsP,GlupczynskiY,NordmannP.EvaluationofaDNAmicroarrayfortherapiddetectionof extended-spectrum β-lactamases (TEM, SHV and CTX-M), plasmid-mediated cephalosporinases (CMY-2-like,DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and carbapenemases (KPC, OXA-48, VIM, IMP and NDM). JAntimicrobChemother.2012;67:1865-9.

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5.PolymyxinresistanceinGram-negativebacilli

ImportanceofdetectionofresistanceRequiredforclinicalantimicrobialsusceptibilitycategorization YesInfectioncontrolpurposes YesPublichealthpurposes Yes

Acquired polymyxin resistance in Enterobacteriaceae has emerged in recent years worldwide.Especially the emergence of plasmid-mediated resistance both in animals, food products andhumansisworrisome,sinceithasagreatpropensityforhorizontaldissemination.Previously,polymyxinresistancewasreportedalwaystobechromosomallymediatedandusuallyrelated to mutations in several genes included in the two- component regulatory system forbiosynthesisof lipidA,and thereby regulationof charge in the lipopolysaccharide (LPS) (1,2). In2015, the first reportsonplasmid-mediated colistin resistanceappeared, andwere related to aplasmid-encodedphosphoethanolaminetransferase,whichaddsaphosphoethanolaminegrouptolipidA.Theresultingeffect isadecreaseof thenetnegativecharge in theLPSandthereby lessinteractionwith thepositively chargedpolymyxins.ThenewresistancedeterminantwasnamedMCR-1 (3). Since then thismechanismof resistancehas beendocumented tobepresent on allcontinents.Oneisolatedatesbacktothe1980sbutitappearsthattheworldwideemergencehashappenedthelastapproximately5years(4).During2016twonewvariantsofMCR-1havebeendescribed–MCR-1.2andMCR-2(5,6).InEuropeaninvasivestrainsofK.pneumoniae,theoverallresistanceratestocolistinis8.6%,andcanbeashighas29%incarbapenem-resistantisolates(7).Itshouldbenotedthatthevariationinratesbetweencountriesisveryhigh,andthatmethodologicalissuescouldcontributetoinflatednumbers.Themajorityof the resistance is thought tobedue tochromosomalmechanisms,buttherehavebeenseveralreportsaboutcarbapenemase-producingEnterobacteriaceaewithMCR-1(4).There are currently no extensively evaluated methods for phenotypic characterization of thedifferent polymyxin resistance mechanisms other than the MIC itself determined by brothmicrodilution(gradientdiffusionanddiskdiffusionareunreliableforthisdrugclass).Recently, itwas discovered that MCR-enzymes are dependent on zinc for their hydrolysis, and that zincchelationcanthereforeinhibittheiractivity(8).InhibitiontestsbasedonEDTAordipicolinicacidare therefore expected. However, the current focus is on detecting polymyxin resistanceregardless of mechanism. Laboratories are advised to always use broth microdilution forsusceptibility testingof colistin, and to always use colistin sulfate (9). Specifically, disk diffusionandgradienttestsshouldnotbeused,astheyareassociatedwithhigh-riskofbothverymajorandmajorASTerrors(10).Recently,acolorimetricmethodwasalsointroduced,butithassofarnotbeen tested for robustness in more than one center (11). If further mechanistic studies arewarranted, thishas tobecarriedoutbymolecularmethods.Thecurrent recommendation is tocarryoutsuchfurthertestingonlyoncolistinresistantisolates.QualitycontrolofcolistinmustbeperformedwithbothasusceptibleQCstrain(E.coliATCC25922orP.aeruginosa ATCC 27853) and the colistin resistantE.coli NCTC 13846 (mcr-1 positive). For

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E.coliNCTC13846,thecolistinMICtargetvalueis4mg/Landshouldonlyonoccasionbe2or8mg/L.5.1References1. GiskeCG.Contemporaryresistancetrendsandmechanismsfortheoldantibioticscolistin,temocillin,fosfomycin,

mecillinamandnitrofurantoin.ClinMicrobiolInfect.2015;21(10):899-9052. Olaitan AO, Morand S, Rolain JM. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in

bacteria.FrontMicrobiol.2014;5:643.3. LiuYY,WangY,WalshTR,Yi LX, ZhangR,etal. Emergenceofplasmid-mediatedcolistin resistancemechanism

MCR-1inanimalsandhumanbeingsinChina:amicrobiologicalandmolecularbiologicalstudy.LancetInfectDis.2016;16(2):161-8.

4. Skov RL,MonnetDL. Plasmid-mediated colistin resistance (mcr-1 gene): threemonths later, the story unfolds.EuroSurveill.2016;21(9).

5. DiPilatoV,ArenaF,TasciniC,CannatelliA,HenriciDeAngelisL,etal.mcr-1.2,aNewmcrVariantCarriedonaTransferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain ofSequenceType512.AntimicrobAgentsChemother.2016;60:5612-5.

6. XavierBB,LammensC,RuhalR,Kumar-SinghS,ButayeP,GoossensH,Malhotra-KumarS.Identificationofanovelplasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016. Euro Surveill. 2016;21(27).

7. European Centres for Disease Prevention and Control (ECDC). Antimicrobial resistance surveillance in Europe2014.AnnualreportoftheEuropeanAntimicrobialResistanceSurveillanceNetwork(EARS-Net)

8. HinchliffeP,YangQE,PortalE,YoungT,LiH,etal.InsightsintotheMechanisticBasisofPlasmid-MediatedColistinResistancefromCrystalStructuresoftheCatalyticDomainofMCR-1.SciRep.2017;7:39392.

9. Bergen PJ, Li J, Rayner CR, Nation RL. Colistin methanesulfonate is an inactive prodrug of colistin againstPseudomonasaeruginosa.AntimicrobAgentsChemother.2006;50(6):1953-8.

10. DafopoulouK,ZarkotouO,DimitrouliaE,HadjichristodoulouC,GennimataV,PournarasS,TsakrisA.ComparativeEvaluationofColistinSusceptibilityTestingMethodsamongCarbapenem-NonsusceptibleKlebsiellapneumoniaeandAcinetobacterbaumanniiClinicalIsolates.AntimicrobAgentsChemother.2015Aug;59(8):4625-30.

11. NordmannP,JayolA,PoirelL.RapidDetectionofPolymyxinResistanceinEnterobacteriaceae.EmergInfectDis.2016;22(6):1038-43.

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6.CarbapenemaseproducingP.aeruginosaandAcinetobacter


Carbapenemase-producing P.aeruginosa and Acinetobacter baumannii-group are common inmanypartsofEurope(1).InP.aeruginosaVIM,mainlyVIM-2,isthedominantenzymeinEuropebutKPCproducershavealsobeennotedinLatinAmericancountries(2).InAcinetobactertheOXAcarbapenemases, mainly OXA-23-, OXA 24/40-, OXA-58-, OXA-143-, OXA-235-like enzymes, arefoundmostcommonly(3).There are currently no specific inhibitors of the class D OXA-carbapenemases and none of theexisting phenotypic methods yield satisfactory results for the detection/identification of thesecarbapenemases in Acinetobacter. Colorimetric tests assays have been tried, but have on thewhole not proved accurate in this genus (4). Acinetobactermay also have carbapenemases ofMBL-type,anditispossiblethattheassayscanworkbetterwiththeseenzymes.ForP.aeruginosatheMBLEtest®aswellasdisk-basedassayshavebeenusedforseveraldecades,butarehamperedbypoorspecificity(5-7).Recently,severalauthorshavealsosuggestedvariousmodifications of the combination disk tests (of either imipenemormeropenem in combinationwithvariousclassBinhibitorycompounds(EDTAorDPA)butthesehavebeenvalidatedinsinglecenter studies, and their robustness in their settings is difficult to ascertain in other (8, 9). Thecolorimetric tests have worked better in P.aeruginosa than in Acinetobacter (10), and areprobably the tests with best proven specificity at themoment. Still, no test seems sufficientlyspecifictobeusedasastand-alonetestwithoutmolecularconfirmation.In general, genotypic approaches should be performed for characterization of putativelycarbapenemase-producing P.aeruginosa and Acinetobacter, but particularly for P.aeruginosasomeoftheabovementionedphenotypicapproachescouldlikelybeofvalueforinitialtesting.ItshouldbenotedthatcarbapenemasetestingwouldbemostclinicallyrelevantinP.aeruginosa,since this species may be carbapenem resistant through multiple chromosomal mechanisms(activeefflux,porinalterationordeficiencies).Contrarily,carbapenemresistanceinAcinetobacterisalmostconstantlyduetoproductionofOXAcarbapenemases.Some suggested control strains are P.aeruginosa NCTC 13437 (VIM-10-producing) andA.baumanniiNCTC13301(OXA-23-producing).NoQC-rangesexistforthesestrains.6.1References1. WalshTR.Emergingcarbapenemases:aglobalperspective.IntJAntimicrobAgents.2010;36Suppl3:S8-14.2. Labarca JA, Salles MJ, Seas C, Guzmán-Blanco M. Carbapenem resistance in Pseudomonas aeruginosa and

AcinetobacterbaumanniiinthenosocomialsettinginLatinAmerica.CritRevMicrobiol.2016;42:276-92.

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3. Higgins PG, Pérez-Llarena FJ, Zander E, Fernández A, Bou G, Seifert H. OXA-235, a novel class D β-lactamaseinvolved in resistance to carbapenems in Acinetobacter baumannii. Antimicrob Agents Chemother.2013;57(5):2121-6

4. Noël A, Huang TD, Berhin C, Hoebeke M, Bouchahrouf W, Yunus S, Bogaerts P, Glupczynski Y. ComparativeEvaluationof Four Phenotypic Tests forDetectionof Carbapenemase-ProducingGram-NegativeBacteria. J ClinMicrobiol.2017;55(2):510-518.

5. LeeK,LimYS,YongD,YumJH,ChongY.EvaluationoftheHodgetestandtheimipenem-EDTAdouble-disksynergytestfordifferentiatingmetallo-β-lactamase-producingisolatesofPseudomonasspp.andAcinetobacterspp.JClinMicrobiol2003;41,4623-4629

6. HansenF,HammerumAM,SkovR,HaldorsenB,SundsfjordA,SamuelsenO.EvaluationofthetotalMBLconfirmkit (ROSCO) for detection of metallo-β-lactamases in Pseudomonas aeruginosa and Acinetobacter baumannii.DiagnMicrobiolInfectDis.2014;79(4):486-8

7. SamuelsenO,BuarøL,GiskeCG,SimonsenGS,AasnaesB,SundsfjordA.Evaluationofphenotypic tests for thedetectionofmetallo-β-lactamase-producingPseudomonasaeruginosa ina lowprevalencecountry.JAntimicrobChemother.2008;61(4):827-30

8. Fournier D, Garnier P, Jeannot K, Mille A, Gomez AS, Plésiat P. A convenient method to screen forcarbapenemase-producingPseudomonasaeruginosa.JClinMicrobiol.2013;51(11):3846-8.

9. Heinrichs A, Huang TD, Berhin C, Bogaerts P, Glupczynski Y. Evaluation of several phenotypicmethods for thedetection of carbapenemase-producing Pseudomonas aeruginosa. Eur J Clin Microbiol Infect Dis.2015;34(7):1467-74.

10. KabirMH,MeunierD,HopkinsKL,GiskeCG,WoodfordN.A two-centreevaluationofRAPIDEC®CARBANP forcarbapenemasedetection inEnterobacteriaceae,PseudomonasaeruginosaandAcinetobacter spp. JAntimicrobChemother.2016;71(5):1213-6.

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7.MethicillinresistantStaphylococcusaureus(MRSA)


7.1DefinitionS.aureus isolateswithanauxiliarypenicillin-bindingprotein (PBP2a/PBP2cencodedbymecA ormecC genes) for which β-lactam agents have low affinity, except for the novel class ofcephalosporinshavinganti-MRSAactivity(ceftarolineandceftobiprole).7.2Clinicaland/orepidemiologicalimportanceMethicillin resistant S.aureus is amajor cause ofmorbidity andmortalityworldwide (1,2). ThemortalityofMRSAbloodstreaminfectionsisdoublethatofsimilarinfectionscausedbymethicillinsusceptible strains due to delayed adequate treatment and inferior alternative treatmentregimens(3).MRSAinfectionsareendemicinbothhospitalsandthecommunityinmostpartsoftheworld.7.3MechanismsofresistanceThe main mechanism of resistance is production of an auxiliary penicillin-binding protein,PBP2a/PBP2c which renders the isolate resistant to all β-lactams except for the novel class ofspecific ‘anti-MRSA’ cephalosporins. These agents have sufficiently high affinity to PBP2a, andprobably also the PBP encoded bymecC, to be active againstMRSA (4). The auxiliary PBPs areencodedbythemecAgeneortherecentlydescribedmecCgene(5).ThemecelementisforeigntoS.aureus and is not present in methicillin susceptible S.aureus. Strains with markedheterogeneous expression of themecA gene and frequently lowMICs of oxacillin hamper theaccuracyof susceptibility testing (5). Some isolates express low-level resistance tooxacillin, butare mecA and mecC negative and do not produce alternative PBPs [borderline susceptibleS.aureus (BORSA)]. These strains are relatively rare and themechanism of resistance is poorlycharacterized,butmay includehyperproductionofβ-lactamasesoralterationofthepre-existingPBPs(6).mecApositiveS.aureus isolateswhichare susceptible tobothcefoxitinandoxacillin (OS-MRSA)duetoinactivationofthemecAhavebeendescribedindifferentpartsoftheworld.Thesestrainsaredifferent from theheterogeneously resistantMRSA,which are alsooxacillin susceptiblebutwhich are resistant to cefoxitin (7, 8). The frequency of such isolates is estimated to beapproximately3%accordingtocombinedconventionalphenotypicresultsandPCRresultspositivefor mecA. Reversion from methicillin susceptibility to methicillin resistance during prolongedantibiotictherapyhasbeenfullydocumentedinonecase(9)butisexpectedtohavehappenedinothercases,therateofsuchreversibleresistanceis,however,unknownatthepresenttime.Suchisolatescanbydefinitiononlybedetectedbymolecularanalysis.Itshouldbenotedthatthisdoesnotapplythatmolecularanalysisshouldbedonewithallstrains,butthephenomenoncanbeofimportance in case of therapeutic failure. If themecA gene is detected randomly or due toscreeningbecauseoftherapeuticfailure,theisolateshouldalwaysbereportedresistant.

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7.4RecommendedmethodsfordetectionofmethicillinresistanceinS.aureusMethicillin/oxacillinresistancecanbedetectedphenotypicallybyMICdeterminationandbydiskdiffusion.AgglutinationcanbeusedtodetectPBP2a,butwillnotreliablydetectPBP2c.GenotypicdetectionwithPCRisreliable.7.4.1DetectionbyMICdeterminationordiskdiffusionThe heterogeneous expression of resistance particularly affects MICs of oxacillin, which canappear susceptible. Cefoxitin is a very sensitive and specific marker of mecA/mecC-mediatedmethicillinresistanceincludinginheterogeneousexpressingstrainsandistheagentofchoice.DiskdiffusionusingoxacillinisdiscouragedandinterpretivezonediametersarenolongerincludedintheEUCASTbreakpointtableduetopoorcorrelationwiththepresenceofmecA.A.Brothmicrodilution:Standardmethodology (ISO20776-1) isusedand strainswith cefoxitinMICs>4mg/L shouldbereportedasmethicillinresistant.B.Diskdiffusion:TheEUCASTdiskdiffusionmethod isused.Strainswithacefoxitin (30µgdisk)zonediameter<22mmshouldbereportedasmethicillinresistant.7.4.2DetectionwithgenotypicandlatexagglutinationmethodsGenotypic detection of themecAandmecC genes by PCR (10, 11) and detection of the PBP2aproteinwithlatexagglutinationkitsispossibleusingcommercialorin-houseassays.PBP2cisnotdetectedbythemajorityofcommercialassays.7.4.3ControlstrainsBelowsomepossiblecontrol strains forphenotypicandgenotypicexperimentshavebeen listedfor convenience.Quality control rangesarenot available for these strains.Usersof commercialmethodsshouldstudythepackageinsertforinformationonwhichcontrolstrainstouse.Table1.ExamplesofcontrolstrainsforMRSA-testing.

Strain Mechanism S.aureusATCC29213 MethicillinsusceptibleS.aureusNCTC12493 Methicillinresistant(mecA)S.aureusNCTC13552 Methicillinresistant(mecC)

7.5References

1. Cosgrove SE, Sakoulas G, Perencevich EN, Schwaber MJ, Karchmer AW, Carmeli Y. Comparison of mortalityassociated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis.ClinInfectDis.2003;36:53-9.

2. deKrakerME,WolkewitzM,DaveyPG, KollerW,Berger J, et al. Clinical impactof antimicrobial resistance inEuropean hospitals: excessmortality and length of hospital stay related tomethicillin-resistant Staphylococcusaureusbloodstreaminfections.AntimicrobAgentsChemother.2011;55:1598-605.

3. de Kraker ME, Davey PG, Grundmann H; BURDEN study group. Mortality and hospital stay associated withresistantStaphylococcusaureusandEscherichiacolibacteremia:estimatingtheburdenofantibioticresistanceinEurope.PLoSMed.2011;8(10):e1001104.

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4. Chambers HF, Deleo FR.Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat RevMicrobiol.2009;7:629-41.

5. García-ÁlvarezL,HoldenMT,LindsayH,WebbCR,BrownDF,etal.Meticillin-resistantStaphylococcusaureuswithanovelmecAhomologueinhumanandbovinepopulations intheUKandDenmark:adescriptivestudy.LancetInfectDis.2011;11:595-603

6. ChambersHF.Methicillin resistance in staphylococci:molecularandbiochemicalbasis andclinical implications.ClinMicrobiolRev.1997;10:781-91.

7. Hososaka Y, Hanaki H, Endo H, et al. Characterization of oxacillin-susceptible mecA-positive Staphylococcusaureus:anewtypeofMRSA.JInfectChemother2007;13:79–86.

8. Andrade-Figueiredo M, Leal-Balbino TC. Clonal diversity and epidemiological characteristics of Staphylococcusaureus:highprevalenceofoxacillin-susceptiblemecA-positiveStaphylococcusaureus(OS-MRSA)associatedwithclinicalisolatesinBrazil.BMCMicrobiol.2016;16:115

9. ProulxMK,PalaceSG,GandraS,TorresB,WeirS,StilesT,EllisonRT3rd,GoguenJD.ReversionFromMethicillinSusceptibility toMethicillin Resistance in Staphylococcus aureus During Treatment of Bacteremia. J Infect Dis.2016;213:1041-8.

10. SteggerM,AndersenPS,KearnsA,PichonB,HolmesMA,EdwardsG,LaurentF,TealeC,SkovR,LarsenAR.Rapiddetection,differentiationandtypingofmethicillin-resistantStaphylococcusaureusharbouringeithermecAorthenewmecAhomologuemecALGA251.ClinMicrobiolInfect.2012;4:395-400.

11. PichonB,HillR, LaurentF, LarsenAR,SkovRL,HolmesM,EdwardsGF,TealeC,KearnsAM.Developmentofareal-timequadruplexPCRassayforsimultaneousdetectionofnuc,Panton-Valentineleucocidin(PVL),mecAandhomologuemecALGA251.JAntimicrobChemother.2012;67:2338-41.

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8. Vancomycin-resistantStaphylococcusaureus


8.1DefinitionTheEUCASTclinicalMICbreakpointforresistancetovancomycininS.aureusis>2mg/L.Inrecentyears vancomycin breakpoints have been lowered, thereby removing the former intermediategroup. However, there are important differences in the mechanism of resistance in VanA-mediated high-level vancomycin resistant S.aureus (VRSA) and non-VanA mediated low-levelresistantisolates.Hence,thetermsvancomycinintermediateS.aureus(VISA)andheteroresistantvancomycin intermediate S.aureus (hVISA) have been maintained for isolates with non-VanA-mediatedlow-levelresistancetovancomycin.TheMICshouldalwaysbedeterminedwhenusingvancomycin to treat a patient with severe S.aureus infection. In selected cases, e.g. whentherapeuticfailureissuspected,testingforhVISAmayalsobewarranted.DuetothecomplexityofconfirminghVISA,antimicrobialsurveillanceisfocusedondetectionofVISAandVRSA.VRSA:VancomycinresistantS.aureus:S.aureusisolateswithhigh-levelresistancetovancomycin(MIC>8mg/L).VISA:VancomycinintermediateS.aureusS.aureusisolateswithlow-levelresistancetovancomycin(MIC4-8mg/L).hVISA:HeterogeneousvancomycinintermediateS.aureus.S.aureusisolatessusceptibletovancomycin(MICs≤2mg/L)butwithminoritypopulations(1in106cells)withvancomycinMIC>2mg/L,asjudgedbypopulationanalysisprofileinvestigation.Itshouldbenotedthatalthoughthesetermsstillremain,alloftheabove-mentionedcategoriesshouldberegardedclinicallyresistant.8.2Clinicaland/orepidemiologicalimportanceThere are no recent investigations of the prevalence of isolates with reduced susceptibility toglycopeptides in Europe. Based on reports from single institutions it is estimated that theprevalenceofhVISAis≤2%ofMRSAinEurope,withVISAbelow0.1%(1).VRSAhasnotyetbeenreportedinEurope(1)andiscurrentlyextremelyrareworldwide(2).TheprevalenceofhVISAmaybeconsiderablyhigherlocally(1),mostoftenassociatedwithspreadofspecificclonallineages(2).The vast majority of isolates with elevated MIC (VISA) or containing resistant subpopulations(hVISA)areMRSA.TheclinicalsignificanceofhVISAhasbeendifficulttodetermineasnowell-controlledprospectivestudies have been performed. However, presence of the hVISA phenotype is believed to beassociatedwith poorer outcome, at least in serious infections (2, 3). It is therefore prudent toinvestigate for the presence of hVISA in bloodstream infections not responding to therapy.RecentlytherehasbeenincreasingevidencethatisolateswithMICsintheupperpartofthewild-

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type range (MIC >1mg/L) are associatedwith poorer outcomeandmaybe linked to increasedmortality, at least in bloodstream infections (3-8). The possible cause of these observations isunclearbutmaybeduetounderexposuretovancomycin(9,10).Furthermore,theinterpretationofthefindingsthesestudiesisconfoundedbydifferencesinMICsgeneratedbydifferenttestingmethods(8,9).

Themechanism of hVISA is complex and detection relies on population analysis (11), which iscumbersome, requires special equipment and needs a high level of technical expertise.Methodology for detection of hVISAwill be outlined, but for surveillance purposes reporting isrestrictedtoVISAandVRSA,whicharetogetherdefinedasisolateswithanMIC>2mg/L.

8.3MechanismofresistanceForVRSA the resistance ismediatedby thevanA gene,exogenouslyacquired fromenterococci.ForbothVISAandhVISAisolatestheresistanceisendogenous(i.e.chromosomalmutations)andthemechanismhighlycomplex,withnosinglegenebeingresponsible.TheVISA/hVISAphenotypeislinkedtoathickeningofthebacterialcellwall,associatedwithhyperproductionofglycopeptidebinding targets. The hVISA phenotype is often unstable in the laboratory, but hVISA have thecapacitytodevelopintoVISAinvivo(2).

8.4Recommendedmethodsfordetectionofvancomycinnon-susceptibleS.aureusDiskdiffusioncannotbeusedto test foreitherhVISAorVISA,butcan likelybeusedto test forVRSA,althoughtherearelimitedstudiestosupportthis(12).

8.4.1MICdeterminationBrothmicrodilutionmethodologyasrecommendedbyEUCAST(ISO20776-1)isthegoldstandard.It should be noted that the resultswith gradient stripmethodsmay be 0.5-1 two-fold dilutionstepshigher than the resultsobtainedbybrothmicrodilution (8,9).TheEUCASTbreakpoint forresistance to vancomycin in S.aureus is MIC >2 mg/L. Isolates with confirmed MICs >2 mg/L(accordingtobrothmicrodilution)shouldbereferredtoareferencelaboratory.hVISAwillnotbedetectedbyMICdetermination.8.4.2TestsdetectingVRSA,VISAandhVISADetection of hVISA has proven difficult and detection is therefore divided into screening andconfirmation.Forscreening,anumberofspecialisedmethodshavebeendeveloped.Confirmationisobtainedbyanalysingthepopulationprofileoftheisolateonagarplatescontainingarangeofvancomycin concentrations (PAP-AUC) (11). This method is technically challenging withoutextensiveexperienceandconsequentlyismostlyperformedbyreferencelaboratories.Amethodbasedonavancomycinandcaseinscreeningagar(13)hasshownhighsensitivityandspecificity,but has so far only been evaluated in one study, and is for that reason cannot yet berecommended.ThefollowingmethodswillalldetectVRSAandVISA,andhavebeenevaluatedinmulticentrestudies(14,15).

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A.Macrogradienttest:Thistestgivesan indicationofreducedvancomycinsusceptibilitybutnotethatthereadingsarenotMICs.Furthermore, thetestdoesnotdifferentiatebetweenhVISA,VISAandVRSA.Thetestmustbesetupaccordingtothemanufacturer’sinstructions.Notealsothattheinoculumishigher(2.0McFarland)thanwithstandardgradienttests,andthegradienttestisappliedonBrothHeartInfusion (BHI) agar instead of Mueller Hinton. Moreover, the test is finally read after 48 h. Apositiveresultisindicatedbyreadings≥8mg/Lforbothvancomycinandteicoplanin,OR≥12mg/Lforteicoplaninalone.

Asbothcriteriaincludeteicoplanin,testingofvancomycincouldbedependentontheresultoftheteicoplanintest.Thealgorithmwouldthenbe:

• Teicoplaninreading≥12mg/L:VRSA,VISAorhVISA

• Teicoplaninreading8mg/L:Testvancomycin.Ifvancomycinreadingis≥8mg/LthenVRSA,VISAorhVISA

• Teicoplaninreading<8mg/L:NotVRSA,VISAorhVISA

B.Glycopeptideresistancedetection(GRD)gradienttest:Testaccordingtothemanufacturer’sinstructions.ThetestisconsideredpositiveiftheGRDstripresultis≥8mg/Lforeithervancomycinorteicoplanin. C.Teicoplaninscreeningagar:AMueller-Hintonplatecontaining5mg/Lteicoplaninisused(14).Severalcoloniesaresuspendedin0.9%salinetoobtainan inoculumwithequivalent turbidity toa2.0McFarlandstandard.Tenmicrolitersofinoculumisdeliveredasaspotonthesurfaceoftheagar,andtheplateincubatedat35°C inair for24to48h.Growthofcoloniesat48h indicatessuggestsreducedsusceptibilitytoglycopeptides.D.ConfirmatorytestingforhVISA/VISA:AnyisolatescreeningpositiveforreducedsusceptibilityandnotidentifiedasVRSAorVISAbyMICdeterminationmaybehVISAandmaybe investigatedbypopulationanalysisprofile-areaundercurve(PAP-AUC)(9),typicallybyreferraltoareferencelaboratory.8.4.3 ControlstrainsBelowsomepossiblecontrolstrainsforphenotypicexperimentshavebeenlistedforconvenience.Quality control ranges are not available for these strains.Users of commercialmethods shouldexaminethepackageinsertforinformationonwhichcontrolstrainstouse.Table1.ExamplesofcontrolstrainsfortestingofvancomycinresistanceinS.aureus.

Strain Mechanism

S.aureusATCC29213 GlycopeptidesusceptibleS.aureusATCC700698 hVISA(Mu3)S.aureusATCC700699 VISA(Mu50)

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8.5References1. Melo-Cristino J, Resina C, Manuel V, Lito L, Ramirez M. First case of infection with vancomycin-resistant

StaphylococcusaureusinEurope.Lancet.2013;382(9888):2052. Howden BP, Davies JK, Johnson PDR, Stinear TP, Grayson ML. Reduced vancomycin susceptibility in

Staphylococcusaureus,includingvancomycin-intermediateandheterogeneousvancomycin-intermediatestrains:resistancemechanisms,laboratorydetectionandclinicalimplications.ClinMicrobiolRev2010;1:99-139

3. VanHalSJ,LodiseTP,PatersonDL.Theclinicalsignificanceofvancomycinminimuminhibitoryconcentration inStaphylococcus aureus infections: a systematic review andmeta-analysis. Clinical InfectiousDiseases 2012; 54:755-771.

4. ChangHJ,HsuPC, YangCC, Siu LK,KuoAJ, et al. Influenceof teicoplaninMICson treatmentoutcomesamongpatients with teicoplanin-treated methicillin resistant Staphylococcus aureus bacteraemia: a hospital basedretrospectivestudy.J.AntimicrobChemother2012,67:736-41.

5. Honda H, Doern CD, Michael-DunneW Jr, Warren DK. The impact of vancomycin susceptibility on treatmentoutcomes among patients with methicillin resistant Staphylococcus aureus bacteremia. BMC Infect Dis. 2011;5:11:335.

6. LodiseTP,GravesJ,EvansA,GraffunderE,HelmeckeM,etal.RelationshipbetweenvancomycinMICandfailureamongpatientswithmethicillin-resistantStaphylococcusaureusbacteremiatreatedwithvancomycin.AntimicrobAgentsChemother2008;52:3315-20

7. RojasL,BunsowE,MunozP,CercenadoE,Rodrigueuz-Creixems,BouzaE.VancomycinMICsdonotpredict theoutcome ofmethicillin-resistant Staphylococcus aureus bloodstream infections in correctly treated patients. J.AntimicrobChemother2012;7:1760-8.

8. Sader HS, Jones RN, Rossi KL, Rybak MJ. Occurrence of vancomycin tolerant and heterogeneous vancomycinresistant strains (hVISA) among Staphylococcus aureus causing bloodstream infections in nine USA hospitals.AntimicrobChemother.2009;64:1024-8.

9. Holmes NE, Turnidge JD, Munckhof WJ, Robinson JO, Korman TM, O'Sullivan MV, Anderson TL, Roberts SA,WarrenSJ,GaoW,HowdenBP, JohnsonPD.VancomycinAUC/MIC ratio and30-daymortality inpatientswithStaphylococcusaureusbacteremia.AntimicrobAgentsChemother.2013Apr;57(4):1654-63.

10. Holmes NE, Turnidge JD, Munckhof WJ, Robinson JO, Korman TM, O'Sullivan MV, Anderson TL, Roberts SA,WarrenSJ,GaoW,JohnsonPD,HowdenBP.Vancomycinminimuminhibitoryconcentration,hostcomorbiditiesandmortalityinStaphylococcusaureusbacteraemia.ClinMicrobiolInfect.2013Dec;19(12):1163-8.

11. WoottonM,HoweRA,HillmanR,WalshTR,BennettPM,MacGowanAP.Amodifiedpopulationanalysisprofile(PAP)methodtodetecthetero-resistancetovancomycininStaphylococcusaureusinaUKhospital.JAntimicrobChemother.2001;47:399-403

12. TenoverFC,Weigel LM,AppelbaumPC,McDougal LK,Chaitram J,McAllister S,ClarkN,KillgoreG,O'HaraCM,JevittL,PatelJB,BozdoganB.Vancomycin-resistantStaphylococcusaureusisolatefromapatientinPennsylvania.AntimicrobAgentsChemother.2004;48:275-80

13. SatolaSW,FarleyMM,AndersonKF,PatelJB.ComparisonofDetectionMethodsforHeteroresistantVancomycin-IntermediateStaphylococcus aureus,with the PopulationAnalysis ProfileMethod as theReferenceMethod. JClinMicrobiol.2011;49:177–183.

14. Wootton M, MacGowan AP, Walsh TR, Howe RA. A multicenter study evaluating the current strategies forisolatingStaphylococcusaureusstrainswithreducedsusceptibilitytoglycopeptides.JClinMicrobiol.2007;45:329-32.

15. Voss A, Mouton JW, van Elzakker EP, Hendrix RG, Goessens W, et al. A multi-center blinded study on theefficiency of phenotypic screening methods to detect glycopeptide intermediately susceptible Staphylococcusaureus(GISA)andheterogeneousGISA(hGISA).AnnClinMicrobiolAntimicrob.2007;6:9.

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9. VancomycinresistantEnterococcusfaeciumandEnterococcusfaecalis


9.1DefinitionEnterococcus faeciumorEnterococcus faecaliswith resistance tovancomycin (VRE) (vancomycinMIC>4mg/L).9.2Clinicaland/orepidemiologicalimportanceEnterococci,especiallyE.faecium,aregenerallyresistanttomostclinicallyavailableantimicrobialagents. Treatment of infections caused by vancomycin-resistant enterococci (VRE) is thereforedifficult, with few treatment options. VRE are known to spread efficiently and persist in thehospital environment, and can colonize many individuals of which only a few may developenterococcal infections (1,2). IsolatesharbouringVanBareusuallyphenotypicallysusceptibletoteicoplanin.Therearetwocasereportsofselectionofteicoplaninresistanceduringtreatmentofenterococci harbouring vanB (3, 4), and recently four cases of therapeutic failure have beendescribed (5), indicating that teicoplanin should be used with caution against enterococci withVanB.TypicalMICvaluesfortheclinicallymostimportantVanenzymesareshowninTable1.Table1.TypicalMICsofglycopeptidesforenterococciexpressingVanAorVanB.9.3MechanismofresistanceClinically relevantresistance ismostoftenmediatedbyplasmid-encodedVanAandVanB ligasesthat replace the terminal D-Ala(nine) in the peptidoglycan with D-Lac(tate). This substitutionreduces thebindingof glycopeptides to the target.VanA-producing strainsexhibit resistance tobothvancomycinandteicoplanin,whereasVanB-producingstrainsusually remainsusceptible toteicoplanin due to lack of induction of the resistance operon. Other Van enzymes of lowerprevalence are VanD, VanE, VanG, VanL, VanM and VanN (6-9), although vanM was recentlyshowntoincreasesignificantlyinChinainE.faecium(10).Additionalenterococcalspecies(i.e.E.raffinosus,E.gallinarumandE.casseliflavus),maycontainvanA, vanBor other van genes encoding enzymes listed above, but these strains are relativelyrare. Chromosomally-encoded VanC enzymes are found in all E.gallinarum and E.casseliflavus

GlycopeptideMIC(mg/L)

VanA VanB

Vancomycin 64-1024 4-1024

Teicoplanin 8-512 0.06-1

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isolates.VanCmediateslow-levelvancomycinresistance(MIC4-16mg/L)butshouldgenerallynotbeconsideredimportantfromaninfectioncontrolpointofview(11).Vancomycin variable enterocci is a term used for VRE where the expression of van genes isphenotypically silenced by genetic rearrangements,whichmay be reversed under glycopeptideselectionpressure(12,13).Moreover, low-MICVRE isatermusedforvanB isolatesthatduetoinitialpoorabilitytobe inducedbyvancomycinhavea lowexpressionofvanBgenesgivingMIClevels below the clinical breakpoint. These low-MIC VRE may increase their MIC to above thebreakpointuponlongerexposuretovancomycin(14).BothVVEandlow-MICVREstrainscanoftenonlybedetectedbymolecularanalysis.Theircurrentprevalenceindifferentgeographicalregionsisunknown.9.4RecommendedmethodsfordetectionofglycopeptideresistanceinE.faeciumandE.faecalisVancomycinresistancecanbedetectedbyMICdetermination,diskdiffusionandthebreakpointagarmethod.Forallthreemethods,itisessentialthatplatesareincubatedforafull24hinordertodetectisolateswithinducibleresistance.All three methods readily detect vanA-mediated resistance. Detection of vanB-mediatedresistanceismorechallenging.MICdeterminationbyagarorbrothdilutionisnotalwaysreliableforVanB,(15-17).OlderreportsshowthatdetectionofvanB-mediatedresistance isproblematicforautomatedmethods(18).Sincethenupdateshavebeenmadetotheautomatedmethods,butmore recent studies on whether the performances of these methods for detection of vanB-mediatedresistancehaveimprovedarelacking.Diskdiffusionwitha5µgvancomycindiskcanbedifficultbutthetestperformswellprovidedtheguidelinesforreadingasspecifiedbyEUCASTarefollowedmeticulously(19).WheninterpretingtheMICordiskdiffusiontestresultsitisimportanttoensurethattheisolateisnotE.gallinarumorE.casseliflavus,whichmaybeerroneously identifiedasE.faeciumdue toapositive arabinose test.MALDI-TOFmass spectrometry is in this context very useful for speciesidentificationofenterococci(20).InsettingswhereMALDI-TOFisnotavailable,theMGP(methyl-alpha-D-glucopyranoside) test or a motility test can be used to distinguish E.gallinarum/E.casseliflavusfromE.faecium(MGPnegative,non-motile).9.4.1MICdeterminationMIC determination may be performed by agar dilution, broth microdilution or gradient MICmethods.Broth microdilution is performed according to the ISO standard 20776-1 as recommended byEUCAST.9.4.2DiskdiffusiontestingFordiskdiffusion,themethodspecifiedbyEUCASTmustbefollowedmeticulously.Inspectzonesfor fuzzyedgesand/ormicrocolonieswith transmitted light. Sharp zoneedges indicate that theisolateissusceptibleandisolateswithsharpzonesandzonediametersabovethebreakpointcanbereportedasvancomycinsusceptible.Isolateswithfuzzyzoneedgesorcolonieswithinthezone(Figure 1) may be resistant regardless of zone size and should not be reported as susceptiblewithout confirmation by MIC determination. In a recent multicenter study the disk diffusion

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methodworked better thanVITEK2 for detection ofvanB-producing enterococci, particularly inlaboratories experienced with ready of fuzzy zone edges (19). Disk diffusion is performedaccordingtotheEUCASTdiskdiffusionmethodologyfornon-fastidiousorganisms.Incubationfor24hisneededinordertodetectresistanceinsomeisolateswithinducibleresistance.

Figure1.ReadingofvancomycindiskdiffusiontestsonEnterococcusspp.

a) Sharpzoneedgesandzonediameter≥12mm.Reportassusceptible.b-d) Fuzzyzoneedgesand/orcolonieswithin thezone.Reportas resistant regardlessofzone

diameter.9.4.3BreakpointagarsBreakpoint agar tests with Brain Heart Infusion agar and 6 mg/L vancomycin are reliable fordetection of vanA- and vanB-positive isolates (19). Breakpoint plates can be obtained fromcommercial manufacturers or made in-house. The breakpoint agar test is performed byapplicationof1x105 -1x106cfu(10µlofa0.5McFarlandsuspension)onBrainHeart infusionagarwith6mg/Lvancomycin.Incubationfor24hat35±1°Cinambientairisneededinordertodetect resistance in some isolateswith inducible resistance.Growthofmore thanonecolony isscoredasapositivetest.9.4.4GenotypictestingDetection of vancomycin resistance by the use of PCR targeting vanA and vanB can also beperformedusingin-houseorcommercialmethods(20-22).9.4.5QualitycontrolBelowsomepossiblecontrol strains forphenotypicandgenotypicexperimentshavebeen listedfor convenience.Quality control rangesarenot available for these strains.Usersof commercialmethodsshouldstudythepackageinsertforinformationonwhichcontrolstrainstouse.Table2.Examplesofcontrolstrainsfortestingofenterococci.

a)

c) d)

b)

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Strain Mechanism

E.faecalisATCC29212 Vancomycin-susceptibleE.faecalisATCC51299 Vancomycin-resistant(vanB)E.faeciumNCTC12202 Vancomycin-resistant(vanA)

9.5References1. MazuskiJE.Vancomycin-resistantenterococcus:riskfactors,surveillance, infections,andtreatment.SurgInfect.

2008;9:567-71.2. TenoverFC,McDonaldLC.Vancomycin-resistant staphylococciandenterococci:epidemiologyandcontrol.Curr

OpinInfectDis.2005;18:300-5.3. Hayden MK, Trenholme GM, Schultz JE, Sahm DF. In vivo development of teicoplanin resistance in a VanB

Enterococcusfaeciumisolate.JInfectDis.1993;167:1224-7.4. KawalecM,GniadkowskiM,Kedzierska J,SkotnickiA,Fiett J,HryniewiczW.Selectionofa teicoplanin-resistant

Enterococcus faecium mutant during an outbreak caused by vancomycin-resistant enterococci with the vanBphenotype.JClinMicrobiol.2001;39:4274-82.

5. Holmes NE, Ballard SA, Lam MM, Johnson PD, Grayson ML, Stinear TP, Howden BP. Genomic analysis ofteicoplaninresistanceemergingduringtreatmentofvanBvancomycin-resistantEnterococcusfaeciuminfectionsinsolidorgantransplantrecipientsincludingdonor-derivedcases,JAntimicrobChemother.2013;68(9):2134-9.

6. Depardieu F, Perichon B, Courvalin P. Detection of the van alphabet and identification of enterococci andstaphylococciatthespecieslevelbymultiplexPCR.JClinMicrobiol.2004;42:5857-60.

7. BoydDA,WilleyBM,FawcettD,GillaniN,MulveyMR.MolecularcharacterizationofEnterococcusfaecalisN06-0364with low-levelvancomycinresistanceharboringanovelD-Ala-D-Sergenecluster,vanL.AntimicrobAgentsChemother.2008;52:2667-72.

8. Xu X, Lin D, Yan G, Ye X, Wu S, Guo Y, Zhu D, Hu F, Zhang Y, Wang F, Jacoby GA, Wang M. vanM, a newglycopeptide resistance gene cluster found in Enterococcus faecium. Antimicrob Agents Chemother.2010;54:4643-7.

9. LebretonF,DepardieuF,BourdonN,Fines-GuyonM,BergerP,CamiadeS,LeclercqR,CourvalinP,CattoirV.D-Ala-d-SerVanN-typetransferablevancomycinresistanceinEnterococcusfaecium.AntimicrobAgentsChemother.2011;55(10):4606-12.

10. ChenC,SunJ,GuoY,LinD,GuoQ,etal.HighPrevalenceofvanMinVancomycin-ResistantEnterococcusfaeciumIsolatesfromShanghai,China.AntimicrobAgentsChemother.2015;59(12):7795-8.

11. RamotarK,WoodsW,LarocqueL,ToyeB.Comparisonofphenotypicmethodstoidentifyenterococciintrinsicallyresistanttovancomycin(VanCVRE).DiagnMicrobiolInfectDis.2000;36:119-24.

12. SivertsenA,PedersenT,LarssenKW,BerghK,RønningTG,etal.ASilencedvanAGeneClusteronaTransferablePlasmid Caused an Outbreak of Vancomycin-Variable Enterococci. Antimicrob Agents Chemother.2016;60(7):4119-27.

13. ThakerMN,KalanL,WaglechnerN,EshaghiA,PatelSN,etal.Vancomycin-variableenterococcicangiverisetoconstitutiveresistanceduringantibiotictherapy.AntimicrobAgentsChemother.2015;59(3):1405-10.

14. GrabschEA,ChuaK,XieS,Byrne J,BallardSA,WardPB,GraysonML. ImprovedDetectionofvanB2-ContainingEnterococcus faecium with Vancomycin Susceptibility by Etest UsingOxgall Supplementation. J. Clin.Microbiol2008;46;1961-4

15. SwensonJM,ClarkNC,SahmDF,FerraroMJ,DoernG,HindlerJ, JorgensenJH,PfallerMA,RellerLB,WeinsteinMP, et al.Molecular characterization andmultilaboratory evaluation of Enterococcus faecalis ATCC 51299 forqualitycontrolofscreeningtests forvancomycinandhigh-levelaminoglycosideresistance inenterococci. JClinMicrobiol.1995;33:3019-21.

16. Klare I, FleigeC,GeringerU,WitteW,WernerG.Performanceof threechromogenicVRE screeningagars, twoEtest® vancomycin protocols, and different microdilution methods in detecting vanB genotype EnterococcusfaeciumwithvaryingvancomycinMICs.DiagnMicrobiolInfectDis.2012;74:171-6.

17. WijesuriyaTM,PerryP,PryceT,BoehmJ,Kay I,FlexmanJ,CoombsGW,IngramPR.LowvancomycinMICsandfecal densities reduce the sensitivity of screening methods for vancomycin resistance in Enterococci. J ClinMicrobiol.2014Aug;52(8):2829-33

18. EndtzHP,VanDenBraakN,VanBelkumA,GoessensWH,KreftD,StroebelAB,VerbrughHA.Comparisonofeightmethodstodetectvancomycinresistanceinenterococci.JClinMicrobiol.1998;36:592-4.

19. HegstadK,GiskeCG,HaldorsenB,Matuschek E, SchønningK, et al. Performanceof the EUCASTdisk diffusionmethod,theCLSIagarscreenmethod,andtheVitek2automatedantimicrobialsusceptibilitytestingsystemfor

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detection of clinical isolates of Enterococci with low- and medium-level VanB-type vancomycin resistance: amulticenterstudy.JClinMicrobiol.2014;52(5):1582-9

20. FangH,OhlssonAK,UllbergM,ÖzenciV.Evaluationofspecies-specificPCR,BrukerMS,VITEKMSandtheVITEK2systemfortheidentificationofclinicalEnterococcusisolates.EurJClinMicrobiolInfectDis.2012;31:3073-7

21. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to thespecieslevelofclinicallyrelevantenterococcibyPCR.JClinMicrobiol.1995;33:1434.

22. Dahl KH, Simonsen GS, Olsvik O, Sundsfjord A. Heterogeneity in the vanB gene cluster of genomically diverseclinicalstrainsofvancomycin-resistantenterococci.AntimicrobAgentsChemother.1999;43:1105-10.

23. GazinM,LammensC,GoossensH,Malhotra-KumarS;MOSARWP2StudyTeam.EvaluationofGeneOhmVanRand Xpert vanA/vanB molecular assays for the rapid detection of vancomycin-resistant enterococci. Eur J ClinMicrobiolInfectDis.2012;31:273-6.

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10.Penicillinnon-susceptible(non-wildtype)Streptococcuspneumoniae

ImportanceofdetectionofresistanceRequiredforclinicalantimicrobialsusceptibilitycategorization YesInfectioncontrolpurposes NoPublichealthpurposes Yes

10.1DefinitionS.pneumoniaeisolateswithreducedsusceptibilitytopenicillin(MICsabovethoseofthewild-type,i.e. >0.06 mg/L) due to the presence of modified penicillin-binding proteins (PBPs) with loweraffinityforβ-lactams.10.2Clinicaland/orepidemiologicalimportanceS.pneumoniae isthemostcommoncauseofpneumoniaworldwide.Morbidityandmortalityarehigh and approximately three million people are estimated to die each year of pneumococcalinfections. Low-level penicillin non-susceptibility is associated with increased mortality whenmeningitis is treatedwith benzylpenicillin (1). In other infection types no increasedmortality isobserved with low-level resistance if higher dosages are given. Many countries carry outvaccinationprogrammesagainstseveralpneumococcalserotypes,andthismayaffectresistancelevelsobservedininvasiveisolates(2).However,penicillinnon-susceptibleS.pneumoniaeremainamajor clinicalproblem fromapublichealthpointof view,although thesemicroorganismsarenot associated with spread in healthcare institutions, unlike many of the other pathogensdescribedinthisdocument.10.3MechanismofresistanceS.pneumoniae contains six PBPs, of which PBP 2x is the primary target of penicillin (3). Thepresence of “mosaic genes“ encoding low-affinity PBPs is the result of horizontal gene transferfromcommensalviridans streptococci (3).The levelofβ-lactamresistancedependsnotonlyonlow-affinitymosaicPBPspresentintheisolate,butalsoonmodificationofthespecificPBPsthatare essential for S.pneumoniae (4). StrainswithMICs of benzylpenicillin in the range 0.12 to 2mg/Lare considered susceptible innon-meningitis infectionswhenahigherdoseofpenicillin isused,whereasformeningitissuchstrainsmustalwaysbereportedasresistant(5).10.4 Recommended methods for detection of penicillin non-susceptibleS.pneumoniaePenicillinnon-susceptibilitycanbedetectedphenotypicallybyMICordiskdiffusionmethods.10.4.1DiskdiffusionmethodThe disk diffusion method with 1 µg oxacillin disks is an effective screening method for thedetectionofpenicillinnon-wildtypepneumococci (6-8).Themethod isverysensitive,but isnothighlyspecificbecausestrainswithzonediametersof≤19mmmayhavevariablesusceptibilitytobenzylpenicillin,andthebenzylpenicillinMICshouldbedeterminedforall isolates thatarenon-wildtypewiththescreeningmethod(8).For β-lactams other than benzylpenicillin the oxacillin zone diameter can be used to predictsusceptibilityasinFigure1.

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Figure1.Screeningforβ-lactamresistanceinS.pneumoniae*Oxacillin1μg<20mm:AlwaysdeterminetheMICofbenzylpenicillinbutdonotdelayreportingof other β-lactams as recommended above and do not delay reporting of benzylpenicillin inmeningitis.

10.4.2ClinicalbreakpointsThe penicillin breakpoints were initially designed to ensure the success of therapy forpneumococcal meningitis. However, clinical studies demonstrated that the outcome ofpneumococcal pneumonia caused bymost strains with elevatedMICs to penicillin and treatedwith parenteral penicillin was no different to that for patients treated with other agents.Consideringmicrobiological,pharmacokineticandpharmacodynamicdata,theclinicalbreakpointsforbenzylpenicillinfornon-meningitisisolateswererevisited(4)andcurrentEUCASTbreakpointsareaslistedinTable1andinthelastversionoftheEUCASTbreakpointtable.Table1.Reportingofbenzylpenicillinsusceptibilityinmeningitisandnon-meningitis.

Indications MIC breakpoint(mg/L)

Notes

S≤ R> Benzylpenicillin(non-meningitis)

0.06 2 Inpneumonia,whenadoseof1.2gx4isused,isolateswithMIC≤0.5mg/Lshouldberegardedassusceptibletobenzylpenicillin.Inpneumonia,whenadoseof2.4gx4or1.2gx6isused,isolateswithMIC≤1mg/Lshouldberegardedassusceptibleto

Disk diffusion test with oxacillin 1 µg disk

Report susceptible to all β-lactam agents for which clinical breakpoints are

available, including those with “Note”, except for cefaclor, which if reported, should be reported as intermediate

Determine the MIC and interpret according to the

clinical breakpoints.

Zone diameter < 20 mm*

Benzylpenicillin* (meningitis)

and phenoxymethylpenicillin

(all indications)

Benzylpenicillin (infections other than meningitis)

Ampicillin, amoxicillin and piperacillin (without and with

β-lactamase inhibitor), cefepime, cefotaxime, ceftaroline,

ceftobiprole and ceftriaxone

Report resistant Determine the MIC and interpret according to the clinical breakpoints. For ampicillin,

amoxicillin and piperacillin (without and with β-lactamase inhibitor) infer susceptibility from

the MIC of ampicillin.

Report susceptible

Oxacillin zone < 8 mm

Zone diameter ≥ 20 mm

Oxacillin zone ≥ 8 mm

Other β-lactam agents

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benzylpenicillin.Inpneumonia,whenadoseof2.4gx6isused,isolateswithMIC≤2mg/Lshouldberegardedassusceptible.

Benzylpenicillin(meningitis)

0.06 0.06

10.4.3QualitycontrolBelowapossiblecontrolstrainforphenotypictestinghasbeenlistedforconvenience.Table2.Exampleofcontrolstrainforbenzylpenicillintesting.

Strain Mechanism

S.pneumoniaeATCC49619 MosaicPBP,benzylpenicillinMIC0.5mg/L10.5References1. Kaplan SL,Mason EO Jr.Management of infections due to antibiotic-resistant Streptococcus pneumoniae. Clin

MicrobiolRev.1998;11(4):628-44.2. DaganR. Impact of pneumococcal conjugate vaccine on infections caused by antibiotic-resistantStreptococcus

pneumoniae.ClinMicrobiolInfect.2009;15(Suppl3):16-20.3. HakenbeckR,KaminskiK,KönigA,vanderLindenM,PaikJ,ReichmannP,ZähnerD.Penicillin-bindingproteinsin

beta-lactam-resistantStreptococcuspneumoniae.MicrobDrugResist1999;5:91-99.4. GrebeT,HakenbeckR.Penicillin-bindingproteins2band2xofStreptococcuspneumoniaeareprimaryresistance

determinantsfordifferentclassesofβ-lactamantibiotics.AntimicrobAgentsChemother1996;40:829-834.5. Weinstein MP, Klugman KP, Jones RN. Rationale for revised penicillin susceptibility breakpoints versus

Streptococcuspneumoniae:Copingwithantimicrobialsusceptibility inaneraof resistance.Clin InfectDis2009;48:1596–1600.

6. Dixon JMS, Lipinski AE, GrahamMEP. Detection and prevalence of pneumococci with increased resistance topenicillin.CanMedAssocJ1977;117:1159-61.

7. Swenson JM,HillBC,ThornsberryC. Screeningpneumococci forpenicillin resistance. JClinMicrobiol1986;24:749-52.

8. JettéLPandCSinave.Useofanoxacillindiskscreeningtestfordetectionofpenicillin-andceftriaxone-resistantpneumococci.JClinMicrobiol1999;37:1178-81.

eucast detection of resistance mechanisms 170711

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