Diversity and regulation of intrinsic

FEMSMicrobiologyReviews,fux043doi:10.1093/femsre/fux043ReviewarticleREVIEWARTICLEDiversityandregulationofintrinsicβ-lactamasesfromnon-fermentingandotherGram-negativeopportunisticpathogensCarlosJuan∗,GabrielTorrens,MarGonzález-NicolauandAntonioOliverServiciodeMicrobiologı́aandUnidaddeInvestigación,HospitalSonEspases-InstitutodeInvestigaciónSanitariadeBaleares(IdISBa),07120Palma,IllesBalears,Spain∗Correspondingauthor:ServiciodeMicrobiologı́aandUnidaddeInvestigación,HospitalSonEspases-InstitutodeInvestigaciónSanitariadeBaleares(IdISBa),CarreteradeValldemossa79,07120Palma,IllesBalears,Spain.Tel:+34699631007;Fax:+34871205000;E-mail:carlos.juan@ssib.esOnesentencesummary:Thisreviewcoversthediversity,regulationandmechanismsleadingtomutationaloverexpressionofintrinsicβ-lactamasesfromnon-fermentingandotherGram-negativeopportunisticpathogens.Therelevantinterplaybetweenβ-lactamaseregulation,peptidoglycanbiologyandfitness/virulenceisalsoaddressed,aswellastheroleofβ-lactamaseregulatorypathwaysastargetstoovercomeβ-lactamresistance.Editor:EhudBaninABSTRACTThisreviewdeeplyaddressesforthefirsttimethediversity,regulationandmechanismsleadingtomutationaloverexpressionofintrinsicβ-lactamasesfromnon-fermentingandothernon-EnterobacteriaceaeGram-negativeopportunisticpathogens.Afterageneraloverviewoftheintrinsicβ-lactamasesdescribedsofarinthesemicroorganisms,includingcirca.60speciesand100differentenzymes,wereviewthewidearrayofregulatorypathwaysoftheseβ-lactamases.TheyincludediverseLysR-typeregulators,whichcontroltheexpressionofβ-lactamasesfromrelevantnosocomialpathogenssuchasPseudomonasaeruginosaorStenothrophomonasmaltophiliaortwo-componentregulators,withspecialrelevanceinAeromonasspp.,alongwithotherpathways.Likewise,themultiplemutationalmechanismsleadingtoβ-lactamaseoverexpressionandβ-lactamresistancedevelopment,includingAmpD(N-acetyl-muramyl-L-alanineamidase),DacB(PBP4),MrcA(PPBP1A)andotherPBPs,BlrAB(two-componentregulator)orseverallytictransglycosylasesamongothers,arealsodescribed.Moreover,weaddressthegrowingevidenceofamajorinterplaybetweenβ-lactamaseregulation,peptidoglycanmetabolismandvirulence.Finally,weanalyserecentworksshowingthatblockingofpeptidoglycanrecycling(suchasinhibitionofNagZorAmpG)mightbeusefultopreventandrevertβ-lactamresistance.Altogether,theprovidedinformationandtheidentifiedgapsshouldbevaluableforguidingfuturestrategiesforcombatingmultidrug-resistantGram-negativepathogens.Keywords:intrinsicβ-lactamase;β-lactamaseinduction;β-lactamasederepression;β-lactamresistance;peptidoglycanrecycling;LysR-typeregulators;two-componentregulators;AmpCINTRODUCTIONForover60years,β-lactamshavebeenthefirstlineofantibiotictreatmentsformanycommunity-andhospital-acquiredinfec-tions,includingthosebymultidrug-resistant(MDR)pathogens,and,despitetheenormouseffortstodevelopnewβ-lactamderivativestoovercomegrowingbacterialresistance,sofarnosinglemoleculeescapesfromhydrolysisbyseveralofthethou-sandsofβ-lactamasesdescribed.Previousexcellentreviewsinthefieldofβ-lactamase-mediatedresistancehavefocusedmainlyonstructuralandfunctionalclassification(Bush,JacobyReceived:6April2017;Accepted:18August2017C©FEMS2017.Allrightsreserved.Forpermissions,pleasee-mail:journals.permissions@oup.com12FEMSMicrobiologyReviewsandMedeiros1995;Walther-RasmussenandHøiby2006;BushandJacoby2010;Philipponetal.2016),plasmid-mediateden-zymes[particularlyextendedspectrumβ-lactamases(ESBLs)andcarbapenemases(Walshetal.2005;FalagasandKarageor-gopoulos2009;Poirel,NaasandNordmann2010;Poirel,PitoutandNordmann2007;Walsh2010;BushandFisher2011;Bush2013;Ghafourianetal.2015)],andintrinsicβ-lactamases(par-ticularlyAmpC)fromEnterobacteriaceae(LindbergandNormark1986;HansonandSanders1999;LivermoreandWoodford2006;Paterson2006;Jacoby2009;FisherandMobashery2014).Thisreviewwillfocusonthediversity,regulationandmechanismsleadingtomutationaloverexpressionofintrinsicβ-lactamasesfromnon-fermentingandothernon-EnterobacteriaceaeGram-negativeopportunisticpathogens.Therearemanyreasonsforthegrowinginterestinthefield.(i)IncreasingclinicalimpactofMDRnon-fermentingopportunisticGram-negativepathogens,includingPseudomonasaeruginosaandAcinetobacterbaumannii,butalsoemergingpathogenssuchasStenothrophomonasmal-tophiliaorAchromobacterxyloxoxidans.(ii)Growingisolationfromhumaninfectionsofunusualnon-fermentingopportunisticGram-negativebacteriaduetopatient-relatedfactors(suchasimmunosuppression)andrecentadvancesintaxonomyandidentificationtools,suchasMALDI-TOFmassspectrometry.(iii)Recentdescriptionofmultiplenovelandcomplexregula-torypathwaysofβ-lactamaseexpressioninnon-fermentingandotheropportunisticGram-negativepathogens.(iv)Growingevi-denceindicatingamajorinterplaybetweenpeptidoglycan(PGN)metabolism,β-lactamaseregulationandvirulence.(v)RecentdelineationoftherapeuticalstrategiesbasedontheblockadeofPGNrecyclingandβ-lactamaseexpressiontocombatinfectionbyMDRopportunisticGram-negativepathogens.INTRINSICβ-LACTAMASESINNON-FERMENTINGANDOTHERGRAM-NEGATIVEOPPORTUNISTICPATHOGENSThepublishedinformationonthepresenceofintrinsicβ-lactamases(withchromosomalcodificationintheclearma-jorityofthecases)innon-fermentingGram-negativespeciesishugeandhasneverstoppedincreasingsincethefirstpublicationsinthe1970s(RichmondandCurtis1974;Sugi-naka,IchikawaandKotani1975).Eventhoughtheoriginofβ-lactamasesisprobablyrelatedwithancestralPGN-modifyingactivities(Morosinietal.2000),theycouldalsohaveanaddi-tionalenvironmentallyrelatedprotectivefunctionagainsttheβ-lactamsproducedbyothermicroorganismsdevelopeddur-ingevolution.Needlesstosay,theimportanceoftheseenzymeshasbeencontinuouslyincreasingthankstothegrowingselec-tivepressureexertedbyhumanandanimalβ-lactamantibiotictreatmentssincethe1950s.Obviously,whentheseenzymesarecodifiedonthegenomesofpotentialopportunisticpathogens,thederivedclinicalconsequencesareclearlyenhanced.Inthissense,thegreatvarietyofpathwaysdeterminingthelevelsofintrinsicβ-lactamasesexpressionconstitutesapowerfulbacte-rialweapontosurvivethetreatmentsthatseverelyandincreas-inglycompromisesourβ-lactamtherapeuticarsenal,andthatweneedtodissectinordertodesignusefulfuturetreatments.AlthoughmanyGram-negativespeciesharbouringintrinsicβ-lactamasesarealmostfullyenvironmental,andhenceshownoclinicalrelevance,knowledgeabouttheregulationoftheex-pressionoftheseβ-lactamasesmustnotbeneglectedasitcouldprovidevaluableinformationforunderstandingthebiologyoftheseenzymesregulationinothermoreclinicallyrelevantspecies.Inthissense,Table1givesanoverviewofthemainnon-fermentingandothernon-EnterobacteriaceaeGram-negativeop-portunisticpathogens,wheresomeinformationabouttheirin-trinsicβ-lactamaseshasbeenpublished.Itshouldbenotedthatnoexperimentalbutonlybioinformaticstoolshavebeenusedtocharacterisesomeofthedisplayedβ-lactamases,andinthese,theclassificationofthefunctionalgroupsdescribedbyBushandJacoby(2010)couldobviouslynotbeestablished.However,inotherworks,theauthorsconsideredthehighlevelofaminoacididentitywithpreviouslydescribedenzymessufficienttolocatetheenzymeinthecorrespondingBushgroup.AsshowninTable1,intrinsicβ-lactamasesmaybelongtoAmblersclassesA,B,CorDandhavemostlybeenreportedtobechromosomallyencodedandcharacteristicoftherespec-tivespecies,butwithsomeexceptions:thepresenceoftheβ-lactamasesCfxAandCSP-1onCapnocytophagasputigenastrainsisnotuniversal(Ehrmannetal.2014),andmoreover,CfxAseemstobemorefrequentlydetectedthanCSP-1,probablyduetotheusualCfxAlocationonaplasmid(Ehrmannetal.2014).InthecaseofMoraxellacatarrhalisBROenzymes,somecontro-versialreportsregardingthechromosomal/plasmidcodificationhavebeenpublished(Steingrube,WallaceandBeaulieu1993;Bootsmaetal.2000).Finally,AeromonascaviaedoesnotharbourthecphAgene,whereasinA.veroniibvsobria,theCepenzymeisnotuniversallyfoundinallthestrains.RegardlessoftheclinicalsignificanceofthemicroorganismsdisplayedinTable1,thevolumeofinformationdealingwiththeregulationoftheirrespectiveβ-lactamasesvariesgreatlyde-pendingonthespecies.Availabledatawillbecollectedinthenextsections,firstdealingwiththemechanismsrelatedwiththeregulation/inductionofβ-lactamaseproductionandlater,withtheunderlyingmechanismsresponsiblefortheirstablehy-perexpression.REGULATIONOFβ-LACTAMASESANDPGNRECYCLINGThissectioncoversthemainmodelsofregulation/induction(mostlyrelatedwithPGNmetabolism)thathavebeendescribedforGram-negativeintrinsicβ-lactamases,classifyingthemintothreesubsections:(i)thoselinkedtoLysR-typeregulators,(ii)regulationdirectlylinkedtoBlrAB-liketwo-componentsystemsand(iii)otherregulatorypathways.Thus,inthissection,wereviewtheregulatorymechanismsdrivingtobasallevelsofβ-lactamasesexpressionandthosemechanismsresponsibleforthereversibleincreaseofexpres-sioncausedbyinducerβ-lactams(orbyalternativeinducers),andinthefollowingsection,wereviewtheknowledgeaboutthemolecularbasisforthestablehyperproductionusuallyselectedbymutationduringtreatments.InTable2,themainfeaturesofthemechanismsthatwillbedissectedfromthispointaresum-marisedtohelpthereader.LysR-typeregulators(AmpR)GeneralmodeldiscoveredinEnterobacteriaceaeanddevelopedinPseudomonasaeruginosaThefirstin-depthstudiesdescribingtheregulationofβ-lactamaseproductionweremadeonthealmostubiquitousGram-negativechromosomalAmpCcephalosporinase,(Jacoby2009)andperformedondifferentspeciesofEnterobacteriaceae,showinganintimatelinkagewithPGNmetabolism(FisherandJuanetal.3Table1.Intrinsicβ-lactamasesfromnon-fermentingandotheropportunisticGram-negativepathogens.β-lactamasenameAmblersBushSpeciesorgeneclassgroupReferencesInducibilityAchromobacterxylosoxidansOXA-114andvariantsD2dDoietal.(2008);Amoureuxetal.(2013);Tragliaetal.(2013)NoAchromobacterruhlandiiOXA-258D2dPapaliaetal.(2013)NDaAcinetobacterbaumanniiADC(AmpC)C1Poirel,BonninandNordmann(2011)NoOXA-51/69-likeD2dfNoAcinetobacterradioresistensOXA-23D2dfFigueiredoetal.(2012)NoAcinetobacterIwoffiiOXA-134D2dfNoAcinetobacterjohnsoniiOXA-211D2dfNoAcinetobactercalcoaceticusOXA-213D2dfNoAcinetobacterhaemolyticusOXA-214D2dfNoAcinetobacterbereziniaeOXA-229D2dfBonninetal.(2012)NoAcinetobacterpittiiADC-12to23(AmpC)C1Beceiroetal.(2004,2009)NoAcinetobacterbaylyiADC-8(AmpC)C1Beceiroetal.(2007)NoAeromonashydrophila,A.caviaeb,A.veroniibvsobriacCphAB3bBakkenetal.(1988)YesCepC2eYesAmpD2dYesBordetellabronchiseptica/parapertussisBOR-1A2aLartigueetal.(2005)NoBurkholderiacepaciacomplex(BCC)dPenAandvariants(PenB-H)A2ePoireletal.(2009)YesAmpCC1HwangandKim(2015)YesB.cenocepaciaBCAM1779(strainJ2315)ANDHoldenetal.(2009)NDBCAM0393(strainJ2315)DNDB.mallei/pseudomallei/thailandensisPenI(PenA,BPS-1)A2beCheungetal.(2002);Tribuddharatetal.(2003);Yietal.(2012);Papp-Wallaceetal.(2013);Randalletal.(2015)NoOXA-42,-43,-57,-59D2dNiumsupandWuthiekanun(2002);Keithetal.(2005)NoCapnocytophagasputigenaCSP-1orSPU-1A2beGuillon,EbandMammeri(2010)NoCfxAA2beEhrmannetal.(2014)NoCaulobactercrescentusMbl1B/CAU-1andvariantsB3aSimmetal.(2001);Docquieretal.(2002)NDChromobacteriumviolaceumNospecificnameC1FarrarandO’dell(1976)YesChromohalobacterspp.HaBLAC1Tokunagaetal.(2004);Araietal.(2015)YesChryseobacteriumgleum/indologenesIND-1toIND-16B3aBellaisetal.(2000b);Wangetal.(2016)NoCIA-1to-4A2beMatsumotoetal.(2012)NDCGA-1A2beBellais,NaasandNordmann(2002b)NoCGB-1B3aBellais,NaasandNordmann(2002a)NoChryseobacteriumpisciumCPS-1B3aGudetaetal.(2015)NoDesulfovibriodesulfuricansDES-1A2beMorinetal.(2002)NDElizabethkingiameningosepticaBlaBB3aBellaisetal.(2000a);GonzálezandVila(2012)YesfCME-1and2D2dNoGOB-1andvariantsB3aNoEmpedobacterbrevisEBR-1B3aBellaisetal.(2002)NoFlavobacteriumjohnsoniaeJOHN-1B3aNaas,BellaisandNordmann(2003)NDFrancisellatularensisFTU-1A2fAntunesetal.(2012)NDFrancisellaphilomiragiaFPH-1A2fTothetal.(2012)NDJanthinobacteriumlividumThin-BB3aRossolinietal.(2001)YesLaribacterhongkongensisHLHK5C1Lauetal.(2005)NoLegionellagormaniiOXA-29D2dFranceschinietal.(2001)NDFEZ-1B3aBoschietal.(2000)NDLegionellapneumophilaLoxAD2dAvisonandSimm(2002)NoLysobacterenzymogenesNospecificnameA2bvonTigerstromandBoras(1990)YesMinibacteriummassiliensisMIN-1A2beBercotetal.(2012)NoMoraxellacatarrhalis/M.nonliquefaciensBRO-1/2A2cWallaceetal.(1989);Eliassonetal.(1992)YesMyroidesodoratimimusTUS-1B3aMammeri,BellaisandNordmann(2002);Al-Bayssarietal.(2015)NDMUS-1,-2B3aND4FEMSMicrobiologyReviewsTable1.Continued.β-lactamasenameAmblersBushSpeciesorgeneclassgroupReferencesInducibilityOchrobactrumanthropiOCH-1C1Higginsetal.(2001);Nadjaretal.(2001)YesPandoraeaspp.OXA-62andOXA-151toOXA-159D2dSchneider,QueenanandBauernfeind(2006);SchneiderandBauernfeind(2015)YesPseudomonasaeruginosaAmpCC1Knott-Hunzikeetal.(1982)YesPoxB(OXA-50)D2dGirlich,NaasandNordmann(2004a)NoPIB-1B/Ce3b/1eFajardoetal.(2014)NoPseudomonasspp.(notP.aeruginosa)AmpCC1www.pseudomonas.comYesPseudomonasotitidisPOM-1B3aBorgiannietal.(2015)NoPsychrobacterimmobilisAmpCC1Feller,SonnetandGerday(1995)YesRalstoniapickettiiOXA-22,D2dNordmannetal.(2000)YesOXA-60D2dfGirlich,NaasandNordmann(2004b)YesRalstoniamannitolilyticaOXA-443(OXA-22-like)D2dSuzukietal.(2015)YesOXA-444(OXA-60-like)D2dfYesRhodobactersphaeroidesNospecificnameC1Baumannetal.(1989)YesRhodopseudomonascapsulatusNospecificnameANDCampbelletal.(1989)NDShewanellaoneidensisBlaA(OXA-54)D2dfPoirel,HéritierandNordmann(2004)YesShewanellaalgaeOXA-55D2dfHéritier,PoirelandNordmann(2004)NDShewanellaxiamenensisOXA-181(andvariantsOXA-48b,OXA-199)D2dfPotron,PoirelandNordmann(2011);Zong(2012)NDShewanellalivingstonensisSh.frigidimarinaSLB-1/SFB-1B3aPoirel,HéritierandNordmann(2005)NDSphingobiumspp.SGM-1A2beLamoureauxetal.(2013)NDStenotrophomonasmaltophiliaL1B3aSainoetal.(1982);Saino,MatsuhishaandMitsuhashi(1984);Avisonetal.(2001);Youenouetal.(2015)YesgL2A2eYesgAmpCC1Crossmanetal.(2008)NDaND:notexperimentallydetermined.bA.caviaedoesnotharbourthecphAgene.cInA.veroniibvsobria,theCepenzymeisnotuniversallyfoundinallthestrains.dBCCincludesthespecies:B.cepacia,B.multivorans,B.cenocepacia,B.vietnamiensis,B.stabilis,B.ambifaria,B.dolosa,B.anthina,B.pyrrociniaandB.ubonensis.eFollowingaphylogeneticanalysis,PIB-1seemstobemorecloselyrelatedtoclassCthantoclassB,butitisfunctionallymorelikeB2(Bushgroup3b)metalloβ-lactamases(MBL),whichcouldindicatethatPIB-1definesanovelfamilyofZn-dependentimipenemases(Fajardoetal.2014).fIncontrasttotherestofspeciesdisplayedinTable1,induciblethroughthetypicalinducerssuchascefoxitinorimipenem,theBlaBfromEl.meningosepticahasbeenshowntobeinducibleinresponsetogrowthphaseorrestrictivenutrientmedia,butnottothecitedβ-lactams.gTheL1andL2β-lactamasesareinduciblethroughalltheβ-lactams,notonlyafterchallengingwiththetypicalonessuchascefoxitinorimipenem.Mobashery2014).Thus,severalstudies,mostlyinCitrobacterfreundiiandEnterobactercloacaeduringthe1980sand1990s,wereusedtodrawupaverydetailedschemeforAmpCregu-lation(Lindberg,LindquistandNormark1987;Lindberg,West-manandNormark1985;Honore,NicolasandCole1986;KorfmanandWiedemann1988;Honore,NicolasandCole1989;KorfmanandSanders1989;Lindquist,LindbergandNormark1989;Dietz,PfeifleandWiedemann1997).Yearslater,itwasproventhatthedescribedmodelswereconservedinothernon-entericspeciessuchasP.aeruginosa,withstudiesinthefirstdecadeofthe21stcenturysignificantlyexpandingtheexistingknowl-edgeprovidedbytheEnterobacteriaceaemodel(Jacobsetal.1994;Normark1995;Jacobs,FrereandNormark1997;Langaee,DargisandHuletsky1998;HansonandSanders1999;Langaee,CagnonandHuletsky2000;Juanetal.2005,2006;FisherandMobashery2014).Figure1showsageneralmodelforthismentionedAmpCregulationschemeinP.aeruginosa,whichisalsoapplicabletootherspeciessuchasBurkholderiacepaciacomplex(BCC).TheGram-negativePGNisbuiltupofchainswithnre-peatsofthedisaccharidemonomerN-Acetyl-Glucosamine(GlcNAc)-N-acetylmuramicacid(MurNAc),connectedtootheridenticalchainsbymeansofstempeptideslinkedtotheMurNacunits.Thestempeptidefromadisaccharidemonomer,alwaysinitiallyapentapeptide(L-Alanine-D-glutamicacid-Diaminopimelicacid-D-Alanine-D-Alanine),connectstoasec-ondstempeptide(fromanotherdisaccharidemonomerlo-catedonadifferentchain),thankstothehighmolecularmasspenicillinbindingproteins(PBPs)(1,2and3),throughtheirtranspeptidaseactivity.ThesePBPscleavetheterminalD-Alafromthefirstpentapeptide(carboxypeptidaseactivity),convert-ingitintoatetrapeptide,anessentialsteptoallowthebindingoftheremainingD-Alafromthenewborntetrapeptidetothediaminopimelicacid(DAP)fromotherpentapeptide(transpep-tidation).Thesebondsallowforthecrosslinkingofdisaccharidechains,whichconstitutetheessentialPGNarchitecture.OncethebasicPGNstructureisbuilt,someotherPBPs,mainlythelowmolecularmassPBPs,arethoughttofinelyshapeit.Inthissense,thesePBPs(4,5and6)exertDD-carboxypeptidaseac-tivitiestoreleasetheterminalD-Alafrompentapeptidesdes-tinednottobecross-linked.Ithasbeenproposedthatthisac-tivityfinelytunesthelevelofcross-linkingamongthedisac-charidechains,giventhat,ifapentapeptideisconvertedintoatetrapeptidebytheactivityofalowmassPBP,thisnewborntetrapeptidecannotbeusedbythehighmolecularmassPBPsfortranspeptidation(Sandersetal.1997;ParkandUehara2008;Ropyetal.2015).Juanetal.5Table2.Summaryofthemainfeaturesofthemostrelevantintrinsicβ-lactamaseregulatorypathwaysreviewedinthiswork.SpeciesPseudomonasaeruginosaBurkholderiacepaciacomplex(BCC)StenotrophomonasmaltophiliaAeromonasspp.Shewanellaoneidensisβ-Lactamase(s)AmpCPenAorvariants(PenB-H)andAmpCL1andL2CphA,CepandAmpBlaA(OXA-54)AdditionalconferredresistancewhenhyperexpressedaCAZ,FEP,PIP,ATMCAZ,ATM,MERNoadditionalresistancethroughstableL1and/orL2hyperproductionCAZ,PIP,CTX,IMP,MERAMP,IMPRegulatory(induction)pathwaymainfeaturesMediatedbyactivator/repressorPGN-derivedfragmentsreachingthecytosolthroughAmpG,andancillaryeffectspromotedbyCreBCresponse.NagZ-dependentpathwayCorregulated.Mediatedbyactivator/repressorPGN-derivedfragmentsreachingthecytosolthroughAmpGCorregulated.Mediatedbyactivator/repressorPGN-derivedfragmentsreachingthecytosolthroughAmpGand/orancillaryeffectspromotedbyCreBCresponse.CombinesNagZ-dependentandindependentpathwaysCorregulated.MediatedbyPGN-derivedfragmentsnotenteringthecytosol(AmpGandNagZindependent)PresumablymediatedbyPGN-derivedfragmentsnotnecessarilyenteringthecytosolthroughAmpG.Additionalintermediariesunknownβ-LactamaseregulatorAmpRPenRAmpR(activatingrolemainlyonL2andweaklyonL1)BlrAUnknownTargetscausingstableβ-lactamasehyperexpression(i)ampD(ampDh2-ampDh3)-inactivatingmutationsampD-inactivatingmutations(i)ampDIinactivatingmutations(i)blrBactivatingmutations(i)InactivatingmutationsinnagZ(ii)dacB-inactivatingmutations(ii)mrcAinactivatingmutations(ii)InactivatingmutationsindacB(ii)InactivatingmutationsinampG(iii)ActivatingmutationsinampR(iii)mltD1inactivatingmutations(iii)InactivatingmutationsinblrY(iii)InactivatingmutationsinmrcA(iv)SltB1,MltBinactivation(iv)ActivatingmutationsinampR(iv)InactivatingmutationsinlpoA(v)Mpl,NuoNinactivation(vi)dacC+pbpG-inactivatingmutationsindacB-defectivebackgroundModel(s)(i,iiandiii)LaboratorymutantsandclinicalstrainsLaboratorymutants(iandii)LaboratorymutantsandclinicalstrainsKnockoutmutantsobtainedfromclinicalstrainsLaboratorymutants(iv,vandvi)Laboratorymutants(iiiandiv)LaboratorymutantsReferencesJuanetal.(2006);Moyáetal.(2009);Zamoranoetal.(2010,2011);Cabotetal.(2012);Tsutsumi,TomitaandTanimoto(2013);Cavallarietal.(2013);Ropyetal.(2015)HwangandKim(2015)OkazakiandAvison(2008);Yangetal.(2009);Linetal.(2011);Huangetal.(2012,2015);Talfanetal.(2013)Niumsupetal.(2003);Tayleretal.(2010)Yinetal.(2014,2015)aAdditionaltotheintrinsicpatternofresistance.AMP:ampicillin;ATM:aztreonam;CAZ:ceftazidime;FEP:cefepime;CTX:cefotaxime;PIP:Piperacillin;IMP:imipenem;MER:meropenem.6FEMSMicrobiologyReviewsFigure1.SchematicrepresentationoftheinterplaybetweenPGNrecycling,ampCregulation(induction)andintrinsicβ-lactamresistanceinP.aeruginosa.Duringregulargrowth,AmpDisbelievedtoplayanimportantroleinallowingtheanhydro-MurNAcpeptides(genericallynamedinducingoractivatingligands,ALs),proceedingfromPGNdegradation,toenterintotherecyclingpathway,finallyacquiringthenatureofUDP-MurNAcpep