What are BetaWhat are Beta--lactamaseslactamases??

Proteins degrading Proteins degrading BetaBeta--lactam’slactam’s

Henrik Hasman Henrik Hasman –– National Food National Food Institute Institute -- DTUDTU

The BetaThe Beta--lactam antibioticslactam antibiotics

Isolated from Isolated from Penicillium Penicillium chrysogenum chrysogenum App. 50 % of the antibiotics used worldwideApp. 50 % of the antibiotics used worldwideThe BetaThe Beta--lactam group is constantly expandinglactam group is constantly expandingIs now being produced semiIs now being produced semi--syntheticallysyntheticallyKills growing cells by interfering with the cellKills growing cells by interfering with the cell--wall synthesiswall synthesisOne of the most important human antibiotics.One of the most important human antibiotics.

Penicillin G Ampicillin (AMP)

PenicillinsPenicillins

Amoxicillin

Cephalosporin C Cefoxitin (FOX)(1. gen. Cephalosporin) (2. gen. cephamycin)

Cephalosporin’s Cephalosporin’s

Cefotaxime (CTX) Cefepime (FEB)(3. gen. Cephalosporin) (4. gen. cephalosporin)

Imipenem (IMP)Carbapenem

Carbapenem’s Carbapenem’s og inhibitorsog inhibitors

Clavulanic acid (CLA)Inhibitor

Gram negativeGram positive

Bakterial cellwall

Peptidoglycan

Narrow spectrum vs. Extended Narrow spectrum vs. Extended spectrum Betaspectrum Beta--lactam’slactam’s

Narrow and moderate spectrum BL’sNarrow and moderate spectrum BL’sPenicillin G and V (PEN)Penicillin G and V (PEN)Methicillin (MET)Methicillin (MET)amoxicillin (AMOX) and ampicillin (AMP)amoxicillin (AMOX) and ampicillin (AMP)CephalotinCephalotin (CEP)(CEP)

Broad and Extended spectrum BL’sBroad and Extended spectrum BL’sCefoxitin (FOX)Cefoxitin (FOX)CefotaximeCefotaxime (CTX) and(CTX) and CeftazidimeCeftazidime (CAZ)(CAZ)CefepimeCefepime (FEB)(FEB)ImipenemImipenem (IMI)(IMI)

WhatWhat areare ESBL’sESBL’s thenthen??

Divided into: Divided into: ampC’sampC’s, , “True “True ESBL”ESBL” and and MetalloMetallo--BL’s.BL’s.

Different affinities to different betaDifferent affinities to different beta--lactam’s.lactam’s.

ESBL and ESBL and plasmidicplasmidic ampC’sampC’s mainly i mainly i Enterobacteriaceae.Enterobacteriaceae.MetalloMetallo--BL mainly i BL mainly i Pseudomonas.Pseudomonas.

First identified 22 years ago (SHVFirst identified 22 years ago (SHV--2).2).

now > 200 different genes.now > 200 different genes.

Approximately 20 different groups.Approximately 20 different groups.

Big difference in homology.Big difference in homology.

Seen in all environments where Extended spectrum betaSeen in all environments where Extended spectrum beta--lactam’s are used.lactam’s are used.

Able to degrade Broad and extended spectrum betaAble to degrade Broad and extended spectrum beta--lactam’slactam’s

Beta-lactamase genes so far….

TEMSHVOXACTX

FOXDHAACCPER

CMYMOXPSEFOX

VEBCMEGESTLA

KLUFECLAT…….

Reduced susceptibility can be caused by upReduced susceptibility can be caused by up--regulated effluxregulated efflux--pumps or defective influx pumps. pumps or defective influx pumps.

Pitfalls:Pitfalls:

E. coliE. coli carries a downcarries a down--regulatedregulated ampCampC betabeta--lactamaselactamase, which, whichcan be activated (upcan be activated (up--regulated) by two mutations. regulated) by two mutations.

ESBL

TEMSHVOXA

CTX-MVEBPERCMESFOFECGES

Plasmidic AmpC’s

CMYACCDHAFOXBILMIRACTKLU

MBL

IMPVIMSPMGIM

Genes in yellow indicate most prevalent types!

The ‘True’The ‘True’ ESBL’sESBL’s::

Often located on transferable plasmids/elementsOften located on transferable plasmids/elements

often found in bacteria lacking a chromosomal often found in bacteria lacking a chromosomal AmpC’sAmpC’s

rarely resistant to inhibitors (results in the ‘synergy effect’)rarely resistant to inhibitors (results in the ‘synergy effect’)

The three different ESBL groupsThe three different ESBL groups

resistant to both 3. and 4. generation resistant to both 3. and 4. generation ceph’sceph’sInducible by betaInducible by beta--lactams.lactams.

Synergy!

> 5mm

CTX

CTX-CLA

The three different ESBL groupsThe three different ESBL groups

AmpC’sAmpC’sOften located on chromosomes (Often located on chromosomes (E. coliE. coli, , CitrobacterCitrobacter, , EnterobacterEnterobacter))

...or on plasmids but originating from chromosomal versions...or on plasmids but originating from chromosomal versions

confers resistance to betaconfers resistance to beta--lactam inhibitors (thus no ‘lactam inhibitors (thus no ‘synergy’synergy’))

confers resistance to cefoxitin (FOX); a 2. gen.confers resistance to cefoxitin (FOX); a 2. gen. cephamycincephamycin))

sensitive to 4. gen. sensitive to 4. gen. ceph’sceph’s (like(like cefepimecefepime (FEB))(FEB))

Not inducible by betaNot inducible by beta--lactams.lactams.

No synergy!

< 5mm

CTX

CTX-CLA

MetalloMetallo betabeta--lactamaseslactamases::

Can be inhibited by metal Can be inhibited by metal chelators chelators (like EDTA)(like EDTA)

mainly found in Pseudomonasmainly found in Pseudomonas

confers resistance to all generations of confers resistance to all generations of ceph’sceph’s

The three different ESBL groupsThe three different ESBL groups

confers resistance to confers resistance to carbaphenems carbaphenems like like ImipenemImipenemrarely found in Enterobacteriaceae.rarely found in Enterobacteriaceae.

Phenotypic differences of the groupsPhenotypic differences of the groupsMBLMBLESBLESBLampCampCPhenotypePhenotype

11Some (especially Some (especially TEM’sTEM’s) are inhibitor resistant!) are inhibitor resistant!

Inhibited by EDTA? NO NO Inhibited by EDTA? NO NO YESYES

Inhibited byInhibited by ClavulanicClavulanic acid? NO YESacid? NO YES11 NONO

Resistance to 3. Gen. Resistance to 3. Gen. Ceph’sCeph’s? ? YES YES YESYES YES YES

Resistance to Resistance to cephamycinscephamycins ((cefoxitincefoxitin)? YES NO )? YES NO YESYES

Resistance to 4. Gen. Resistance to 4. Gen. Ceph’sCeph’s? NO YES ? NO YES YESYES

Resistance to Resistance to carbaphenem’scarbaphenem’s? NO NO YES? NO NO YES

How to detect ESBL I?How to detect ESBL I?PhenotypicallyPhenotypically::–– Combination disk methodCombination disk method

–– Double disk methodDouble disk method

–– EE--testtest–– MIC testMIC test

CTXCTX

CTXCTXCLACLA

15 mm15 mm

22 mm22 mm

CeftazidimeCeftazidime 0.250.25--128 µg/ml128 µg/mlCeftazidimeCeftazidime++clavclav. 0.25/4. 0.25/4--128/4 µg/ml128/4 µg/mlAndAndCefotaximeCefotaxime 0.250.25--64 µg/ml64 µg/mlCefotaximeCefotaxime++clavclav. 0.25/4. 0.25/4--64/4 µg/ml64/4 µg/ml

(Confirmatory testing requires use of both(Confirmatory testing requires use of bothcefotaximecefotaxime andand ceftazidimeceftazidime alone and in alone and in combination withcombination with clavulanicclavulanic acid).acid).

CefpodoximeCefpodoxime 4 µg/ml or4 µg/ml orCeftazidimeCeftazidime 1 µg/ml or1 µg/ml orAztreonam 1 µg/ml orAztreonam 1 µg/ml orCefotaximeCefotaxime 1 µg/ml or1 µg/ml orCeftriaxone 1 µg/mlCeftriaxone 1 µg/ml

(The use of more than one (The use of more than one antimicrobial agent for antimicrobial agent for screening will improve the screening will improve the sensitivity of detection).sensitivity of detection).

Antimicrobial Antimicrobial concentrationconcentration

Phenotypic Confirmatory TestPhenotypic Confirmatory TestInitial Screen Initial Screen TestTest

MethodMethod(CLSI 2007)(CLSI 2007)

How to detect ESBL II?How to detect ESBL II?

BreakpointsBreakpoints (CLSI): (CLSI): Ceftazidime: Ceftazidime: SS: : ≤≤ 8; 8; II: 32;: 32; RR: : ≥≥ 3232Cefotaxime: Cefotaxime: SS: : ≤≤ 8; 8; II: 16: 16--32; 32; RR: : ≥≥ 6464

BreakpointsBreakpoints (EFSA): (EFSA): Cefotaxime: Cefotaxime: RR: : ≥≥ 0.50.5

SynergySynergy (CLSI):A (CLSI):A ≥≥ 3 3 twotwo--fold fold reduction reduction in MIC (e.g. from 8 to 1 µg/ml) in MIC (e.g. from 8 to 1 µg/ml)

Screening for ESBL at the CRL

Secondary : Ceftazidime (CAZ - 3. gen.)screening CAZ + CLA (CAZ-CLA + inhibitor)(Disc’s) Cefotaxime (CTX - 3. gen.)

CTX + CLA (CTX-CLA + inhibitor)Cefoxitin (FOX – 2. gen. cephamycin)Cefepime (PER - 4. gen.)

Primary screening : Ampicillin(MIC) Amoxicillin + clavulanic acid

Cephalothin (1. generation cephalosporin)

Cefpodoxime (3. generation cephalosporin)

Ceftiofur (3. generation cephalosporin)

FEB CTX

CAZ

CAZ+CLA

CTX-CLA

FOX

ESBL-tablet assay

FEB

FOX

CTX

CTX-CLACAZ

CAZ-CLA

ESBL ampC

> 5mm

Synergy!

> 5mm

FEB

FOX

CTX

CTX-CLACAZ

CAZ-CLA

< 5mm

No synergy!

< 5mm

BUT BUT BUT…….

FEB

FOX

CTX

CTX-CLACAZ

CAZ-CLA

And one strain can easily have more than onebeta-lactamase!

Different beta-lactamases can have differentaffinity towards different Beta-lactams!

CTXCTX--MM--99 CTXCTX--MM--1414 CTXCTX--MM--11

Strain S 1.3Strain S 1.3 Strain S 1.4Strain S 1.4 Strain S 1.6Strain S 1.6

Table 3Table 3

Future project on phenotypic detection and Future project on phenotypic detection and characterzationcharacterzation of ESBLof ESBL

≈≈250 well250 well--characterized characterized E.E. colicoli and and SalmonellaSalmonella isolatesisolates

50 AMP50 AMPSS E.E. colicoli and 50 AMPand 50 AMPSS SalmonellaSalmonella isolatesisolates25 AMP25 AMPRR E.E. colicoli and 25 AMPand 25 AMPRR SalmonellaSalmonella isolatesisolates2525 ampCampC upup--regulated regulated E.E. colicoli75 75 genogeno--typed ESBL resistant typed ESBL resistant E.E. colicoli and and SalmonellaSalmonella

8 relevant veterinary and human8 relevant veterinary and human cephalosporincephalosporin’’s s

LongLong--rang MIC testing as well as disc diffusion testing rang MIC testing as well as disc diffusion testing

Two different laboratories (FOODTwo different laboratories (FOOD--DTU and CIDCDTU and CIDC--LelystadLelystad))

Future project on phenotypic detection and characterzation of ESFuture project on phenotypic detection and characterzation of ESBLBL

Cefoperazone [0.06Cefoperazone [0.06--128]128]Cefotaxime Cefotaxime [0.015[0.015--32]32]Ceftiofur Ceftiofur [0.06[0.06--128]128]Ceftriaxone Ceftriaxone [0.015[0.015--32]32]Cefquinome Cefquinome [0.015[0.015--32]32]Cefuroxime Cefuroxime [0.12[0.12--128]128]Cefpodoxime Cefpodoxime [0.06[0.06--64]64]Ceftazidime [0.03Ceftazidime [0.03--32]32]

MIC (TREK)MIC (TREK) Disc’sDisc’s (BD)(BD)

75 µg75 µg30 µg30 µg30 µg30 µg30 µg30 µg30 µg30 µg30 µg30 µg10 µg10 µg30 µg30 µg

REQUEST: Please inform us, in case you have ESBL resistantREQUEST: Please inform us, in case you have ESBL resistantE. ColiE. Coli or or SalmonellaSalmonella isolates with KNOWN resistance genes!isolates with KNOWN resistance genes!

–– PCRPCR–– Southern blotting (RLB)Southern blotting (RLB)–– MicroarrayMicroarray–– Cloning and sequencingCloning and sequencing

Genotypic detection of the groupsGenotypic detection of the groups

S / I / RS / I / RSS≥≥ 32/1632/161 / 21 / 2>4>4Type 2Type 2(n=9)(n=9)

S / I / RS / I / RRR4/2 4/2 16/816/8=8 / =8 / ≥≥88>4>4Type 1Type 1(n=11)(n=11)

CeftazidimeCeftazidime(Disc’s)(Disc’s)

CefotaximeCefotaxime(Disc’s)(Disc’s)

AmoxAmox / / clavclav..(µg/ml)(µg/ml)

CeftiofurCeftiofur(µg/ml)(µg/ml)

CefpodoximeCefpodoxime(µg/ml)(µg/ml)

E. ColiE. Coli from Danish veterinary submissions in 2006from Danish veterinary submissions in 2006

Type 1: ”True Type 1: ”True ESBL’sESBL’s” (CTX” (CTX--M1, CTXM1, CTX--MM--2 2 or or CTXCTX--MM--9)9)

Type 2: Type 2: Chromosomal Chromosomal ampC ampC upup--regulators.regulators.