60 yo man post lung transplant. Developedurinary tract infection and sepsis. Blood culturesgrew E. coli. Serum creatinine 200.Resistant• Amp• Cef• Ceftriax• Cipro• Septra• Gent• TobraSensitive• Pip-Tazo• Carbapenem• Amikacin• Nitrofurantoin
Treated with Pip/tazo and relapsed• Sept 28• Oct 21• Nov 15• Dec 19• Feb 13Feb/Mar treated with meropenem (rash) thenamikacin x 14 days with permanenteradication
Bottom lineESBL (E. coli and Klebsiella) class Ashould be treated with a carbapenem IFa beta lactam antibiotic must be used.(and the ESBL is not a carbapenemase)SPICE organisms should be treated witha carbapenem if other antibiotics cannotbe used
β- lactam antibioticsCarbapenem
β-lactam resistanceGram positive• S. aureus beta lactamase• Enterococcus occ. BlaGram negative• Neisseria PBP changestogether with permeabiltiychanges or efflux pumpsPBP changesnovel (PBP2a or PBP5)Mutation or cassette exchangeBeta lactamases• Modulated by permeabiltiyand efflux
OMIMActivity of beta-lactam antibiotic is a balance between:1. Ability of drug to bind to and inactivate PBPs2. Permeability of OMP porins for drug3. Amount of β-lactamase4. Activity of β-lactamase on specific antibioticHighly permeable antibiotic with relatively low susceptibility to β-lactamase will swamp it and inhibit organism.
Intrinsic Susceptibility/resistanceSalmonellaProteus mirabilisE coliShigellaAmp SAmp SAmp SAmp SChromosomal AmpCnot inducibleKlebsiella sp. Amp R, Cef S Chrom. SHV BlaSPICE Amp R, Cef R, Cefox R ChromCeftriax SAmpC ind.Stenotroph. Carbapenem R ChromMetallo Bla
Evolution of ESBLs• E coli became resistant to ampicillin by acquiringplasmids with TEM beta lactamase• 3 rd generation cephalosporins were designed to getaround this. Heavy use of these drove ESBL.• TEM mutants arose resistant to these new drugs• SHV plasmid capture from Klebsiella chrom.• CTX-M plasmid capture from Kluyvera sp. Chrom• Amp C plasmids capture from Enterobacter etc.
Bla and ESBL timelineNMD-12010
β-lactamases Class C or 1 Class A or 2GeneticAmpCChromosome(SPICE)TEM, SHVCTX-MPlasmidRegulation Inducible ConstitutiveSpectrumCephalosporinsand cephamycinsPenicillins,caephalosporinsNOT cephamycinsInhibited by 10mMClavulanic acid -- +Inhibited by 1.0mMaztreonam + --
• E.coli and Klebsiella become resistant to betalactams by acquiring plasmids with thegenes.• SPICE organisms become resistant bychanging from inducible AmpC to constitutivehigh level production, but can also acquireplasmid mediated ESBLs
Gln 39TEM-3 a mutant with extended spectrumfor 3 rd generation cephalosporinsTEM-1Lys 39Glu104Gly238TEM-2Lys 39Glu104Gly238TEM-3Lys104Ser238
Klebsiella MIC with Extended Spectrum betalactamase(ESBL)These ones are TEM mutantsEnzyme Ctx Czid Mox Aztr ImpCtx-1 8-256 8-64 1-4 8-32 0.12-1Caz-1 4-8 32-64 1-2 4-8 0.12TEM-2 0.12 0.5 0.25 0.12 0.12No enz 0.06 0.12 0.12 0.03 0.12+ClavulinicCtx-1 .06-0.5 0.12 1-2 0.12Caz-1 0.12-0.5 0.12 1-2 .06 0.12TEM-2 0.12 0.25 0.25 0.12 0.12
Zone enhancement of clavulanic acid (amox/clav) disc withESBL (R ) compared with wild type TEM or SHV (L) This isused to confirm Cefpodoxime screen by showing that aclavulanic acid-inhibitable b–lactamase is being producedCeftazClavZone enlargement due to Blainhibition by Clavulanic acid
Plasmid-mediated b-lactamases in Gramnegative BacilliClass A:• Inhibited by clavulanic, sulbactam, tazobactam• Cephamycins active• TEM, SHV, CTX-M, KPC-2• CTXM becoming established as dominant worldwide• CTX15 ST131 becoming a dominant clone across world (20-60% OFESBL in Canadian centres)AmpC:• Not inhibited by clavulin etc• Cephamycins not active• Resistant to cephalosporins, cephamycins and Pip/tazo• CMY, MOX, FOX etc• Less likely to be multidrug-resistantCarbapenems active on both
Prevalence of ESBL-producers1999-2000 CanadaE. Coli 0.26%Klebsiella sp 0.81%Predominantly Urine followed byblood wound/respAbout70% Septra R50% Cipro R50% Gent RICU isolates Canada 2005E. Coli 3.7%Klebsiella sp. 1.8%Similar sources78% Cipro R55% Septra R30% Gent R
Break point selection• Pressure from industry to select higherbreak points• Pharmacodynamics increasingly beingused to guide• With β-lactams also consider innoculumeffect: cephalosporins and betalactamase inhibitor combinations havelower MICs at 10 5 than at 10 7
Pip/Tazo vs Carbapenem• Evidence for using carbapenem is mostlyanecdotal• No DECENT studies comparing pip/tazo withcarbapenem• Trend to increase mortality with pip/tazo BUTnumbers too small• Need comparable patients re site of infection,source control, co-morbidity and organisms.
Sequential ESBL outbreaks in an ICU where“decontamination” with 3 rd generationcephalosporins was practicedSHV-2 typeCTX-1SHV-2N D J F M A M J J A S O N D J F M A19851986 1987
GeneGram negative β-lactamasesClass C or 1 Class A or 2AmpCChromosome“SPICE”TEM, SHV,CTX-MPlasmidRegulation Inducible ConstitutiveSpectrum Cephalosporinases PenicillinasesInhibited by 10µMClavulanic acid -- +Inhibited by 1.0µMaztreonam + --
AmpC β-lactamaseCommon in:• Serratia• Providencia• Indole positive Proteus• Citrobacter sp• Enterobacter sp. esp E.cloacae(Pseudomonas,Morganella)• Usually in individualpatientsAmpC β-lactamase is usuallyproduced at very lowlevels in these organismsAmpC constitutive mutants• Resistance to all third gen.cephalosporins.• Resistance to Piperacillin andPip/tazo• Sensitive to carbapenemsunless concomitant OMPmutation
In uninduced state, little β-lactamase produced.Antibiotic binds to PBPs andinhibits/kills organism.In induced stateperiplasmic β-lactamasehydrolyses antibioticbefore it binds to PBPand no inhibition/killingoccurs.SignalInduced stateRampCampC beta-lactamases
Peptidoglycan structureN-acetyl glucosamineMuramic acidTetrapeptidePentaglycine bridge
Model of ampCregulation based onrelative amounts ofmurein intermediates incytoplasm.ampC: beta-lactamasestructural geneAmpG: Membrane importerfor peptidoglycanmetabolites fromperiplasmAmpD: Removes tripeptidefrom N acetylmuramic acidAmpR: Represses oractivates ampCtranscription
Beta-lactamase inductionConstitutive mutantInduction below MICLeakyInduction above MICTightly regulated
Beta-lactams that induce betalactamases1. Strong inducersbelow MIC• Destroyed if labile• Not active exceptimipenem• Ampicillin• Cefoxitin• Imipenem2. Weak inducers• May select stablyderepressed ampCmutants• 3 rd gen. cephalosporins• Carboxy penicillins• Ureidopenicillins
Induction of beta-lactamase by cefoxitin cutsof zone of inhibition around cefotaxime discCefoxitinCefotaxime• Cefoxitin induces ampCbelow MIC and there is nozone of inhibition• Cefotaxime induces aboveits MIC• Zone is large, but is cut offin area next to cefoxitin discwhere cefoxitin has inducedBla expression
AmpC induction MICs
Bottom lineESBL (E. coli and Klebsiella) class A should betreated with a carbapenem IF a beta lactamantibiotic must be used. (and the ESBL is not acarbapenemase)SPICE organisms should be treated with acarbapenem if other antibiotics cannot be usedOther drugs to consider: cipro, septra, tigecycline,doxycycline, aminoglycosides, cefipime
Amp C in E. coli• 3 rd generation cephalosporin resistance in E. coli and Klebsiellais usually a marker for TEM and SHV plasmid mediated β-lactamase mutants.• THESE mutants identified by the characteristic changes in zonesize seen in double disc diffusion test with clavulin; oftensensitive to cefoxitin (unless concomitant porin change)• ampC gene present in E. coli but ampR is not.• AmpC phenotype uncommon, but can be conferred bypromoter mutations• Occasionally, ampC is carried on a plasmid
Gram negative carbapenemasesClass BExampleGeneKPC, VIM, NDM-1Plasmid(chromosome)RegulationSpectrumConstitutiveCarbapenemase andCephaseInhibited by EDTA +Inhibited by 1.0mMaztreonam --
Plasmid mediated CarbapenemasesKPC-1, KPC-2– Class A– Started in New York due to widespread carbapenem use– Resistant to 3rd gen cephalosp.VIM, IMP– Metallo β−lactamase– Pseudomonas Calgary– Enterobacteriaceae GreeceBla NDM-1– Metallo β−lactamase– Enterobacteriaceae– Important in India, Pakistan and returnees
Arch Int Med 2005; 165: 1430• 45% of all K. pneumoniae isolates from 11 Brooklinehospitals produced ESBLs• Carbapenems were the agents of choice for treatingthese infections• KPC-2 ESBL found IN 3.3% OF Brooklyn ESBLproducingK. pneumoniae• Also caused an outbreak in NYC hospital involving 24patients• This strain was distinct from Brooklyn strains
Carbapenemase detection• Resistant to most cephalosporins• Ertapenem MIC >1• Imipenem-EDTA synergy discs• MBL Etest
Modified Hodge Test• 10µgm Imipenem disc• MH agar• E. coli 1:10 of .5McFarland on plate• Cross streak with teststrain and look forgrowth around streakinside inhibition zone.
MBL EtestRatio of 8 or moreSensitivity 96%Specificity 95%Compared to genotype
Bla NDM-1• Metallo β−lactamase• Indian subcontinent• Travellers import toUK• Resistant to almosteverything exceptcolistin and tigecycline
How did TEM beta-lactamase get into Haemophilusspecies and Neisseria gonorrhoeae?Evidence suggests that the TEM gene whichresides on a 5kb transposon named TnA wasintroduced to Haemophilus species andrescued by transposition onto a plasmidwhich is fairly commonly found in throatisolates of Haemophilus parainfluenzae.
Chromogenic Cephalosporin (Nitrocefin)positive on (R )
Transposon TnAEnteric betalactamaseplasmidE. coliPLASMIDSOBSERVEDTransfer not observedH. speciesHaemophilus crypticplasmidH. speciesNeither plasmids nortransfer observedBeta-lactamase plasmidtransferred from ENTERICbacterium to H. species byconjugation(mobilization)?CONCLUSION: TnARESCUED BYINSERTION INTOHAEMOPHILUS SP.CRYPTICENDOGENOUSREPLICON.H. parainfluenzaeH. sp.Enteric plasmidrestricted or fails toreplicate. TnArescued bytransposition ontocrypticendogenousreplicon inHaemophilus sp.
Many strains of H. parainfluenzae isolated from URT and GUtract have small plasmids which are homologous to the H.ducreyi and N. gonorrhoeae beta-lactamase plasmids exceptthat they do not have any TEM transposon sequences