COL Helen Viscount, PhD, D(ABMM)LTC Steven Mahlen, PhD, D(ABMM)

The Beta-Lactamase Family: Classification, Detection, and

Interpretive Criteria

Transplant patient Extremely resistant

Klebsiella pneumoniae recoveredSensitive only to colistin

and gentamicin Patient put in isolation Isolate transmitted to 10

other patients Outcomes:

4/5 with bacteremia died1 other died2 with renal failureOnly 4/11 discharged

without renal failure

Ampicillin: R Pip/tazo: R Ceftazidime: R Ceftriaxone: R Cefepime: R Imipenem: R Meropenem: R Aztreonam: R Amikacin: R Tobramycin: R Trimeth/sulfa: R Fluoroquinolones: R Gentamicin: S Colistin: S

Nursing home resident83 years oldPneumonia

Admitted to ICUStarted on ceftriaxone

and levofloxacinBlood cultures +

K. pneumoniaeBased on sensi’s:

No more levoKept on ceftriaxone

Patient got worseHad to be ventilated

Ampicillin: RPip/tazo: SCefazolin: RCeftazidime: ICeftriaxone: SCefepime: S Imipenem: SAztreonam: STobramycin: STrimeth/sulfa: RLevofloxacin: ICiprofloxacin: IGentamicin: S

ObjectivesAt the end of this

workshop the attendee should be able to distinguish ESBL positive from carbapenemase-producing bacteria

At the end of this workshop the attendee should be able to describe a method to screen for ESBLs

At the end of this workshop the attendee should be able to interpret the results of the modified Hodge Test

Beta-lactam antibioticsPenicillins

AmpicillinAmoxicillinPiperacillin

Cephalosporins (generations)1st gen: cephalothin2nd gen (cephamycins): cefoxitin, cefotetan3rd gen: ceftazidime, cefotaxime, ceftriaxone4th gen: cefepime

Beta-lactam antibioticsMonobactam: aztreonamCarbapenems:

ImipenemMeropenemErtapenem

InhibitorsSulbactam (ampicillin/sulbactam: Unasyn)Tazobactam (piperacillin/tazobactam: Zosyn)Clavulanate (amoxicillin/clavulanate:

Augmentin)

Mechanisms of ResistanceAltered target (Gram negative/positive)Altered permeability (Gram negative)Production of inactivating enzymes (Gram

negative/positive)

Gram-negative cell Gram-positive cell

Outer membrane

PeptidoglycanPeptidoglycan

PenicillinBinding proteins(PBPs)

Inner (cytoplasmic) membrane

Alteration of TargetResistance to -lactams via altered

penicillin-binding proteins (PBPs)MRSA

Vancomycin resistance in enterococciFluoroquinolone resistance

Altered PermeabilityPassive diffusion of Gram-negative cell wallMutate outer membrane proteinsActive efflux

Active Efflux

Production of Inactivating EnzymesChloramphenicol acetyltransferaseAminoglycoside-modifying enzymes-Lactamases

-LactamasesWell over 340 different enzymesExtended spectrum -lactamases (ESBLs)AmpC -lactamases

ChromosomalPlasmid-mediated

Carbapenemases

-LactamasesFirst -lactamase identified: AmpC beta-

lactamase1940, Escherichia coli1940, penicillinase, Staphylococcus aureus

First plasmid-mediated -lactamase: TEM-11965, Escherichia coli, Greece

-Lactamase Activity

C C

C N

H H

R-CONH

S

COOH

CH3

CH3

O

Enzyme-Ser-OH

-lactam

-Lactamase Activity

C C

C N

H H

R-CONH

S

COOH

CH3

CH3

O

HO

Ser

Enzyme

HOH

LL

LL

LL

LL

LLLL

LL

LL LL-lactamaseproduction

Types of Beta-LactamasesESBLsAmpCsCarbapenemases

ESBLs

ESBLsExtended-spectrum beta-lactamases

(ESBLs) are mutant enzymes with a broader range of activity than their parent molecules

They:Hydrolyze 3rd and 4th gen cephalosporins and

aztreonamDo not affect cephamycins (2nd gen ceph) or

carbapenemsRemain susceptible to beta-lactamase

inhibitors

ESBLsThe most common plasmid-mediated ß-

lactamases in Enterobacteriaceae are TEM-1, TEM-2, and SHV-1TEM: Escherichia coli

Named after first patient with a urinary tract infection that was not treatable with ampicillin

Her name: TemorinaSHV: Klebsiella pneumoniae

“Sulfhydryl variant”; amino acids in the enzyme that cross-link with other molecules

“Classical” ESBLs are derived from TEM and SHV enzymes

“Non-classical” ESBLs are derived from enzymes other than TEM or SHV

Classical ESBLsPrimarily found in E. coli and Klebsiella spp.Differ from their parent TEM or SHV

enzymes by only 1-4 amino acids>100 TEM- or SHV-derived beta-lactamases

have been described – most are ESBLs

Non-classical ESBLsMany described, but less common than

classical ESBLsCTX-M

Found in multiple genera of EnterobacteriaceaePreferentially hydrolyze cefotaximeU.S., Europe, South America, Japan, Canada

OXAMainly in P. aeruginosaPrimarily hydrolyze ceftazidimeFrance, Turkey

ESBL EpidemiologyESBLs first appeared in Europe in the mid-

1980sWorldwide, but prevalence varies widely

geographically and between institutionsU.S. national average for ESBLs in

Enterobacteriaceae ~3%

ESBL EpidemiologyESBL producers especially prevalent in

ICUs and long term care facilitiesBecoming more widespread in the community

alsoHave been associated with outbreaks

Typically arise in ICUPlasmid transfer between GNRsOrganism transfer between patients

Control of outbreaksInfection control practice – isolationRestriction of 3rd and 4th generation cephalosporinsAntimicrobial cycling

Clinical Significance

Despite appearing susceptible to one or more penicillins, cephalosporins, or aztreonam in vitro, the use of these agents to treat infections due to ESBL-producers has been associated with poor clinical outcome

Clinical SignificanceESBL genes are often carried on

plasmids that also encode resistance to multiple classes of antimicrobialsAminoglycosides, FluoroquinolonesTrimethoprim/Sulfamethoxazole

Treatment experience is largely based on classical ESBL producersCarbapenemsß-lactam/inhibitor combinations

Typical ESBL Susceptibility ProfileAmp: RPiperacillin: RPip/tazo: SCefazolin: RCefoxitin: SCeftazidime: SCeftriaxone: RCefepime: RAztreonam: SImipenem/

meropenem: S

Amp: RPiperacillin: RPip/tazo: SCefazolin: RCefoxitin: SCeftazidime: RCeftriaxone: RCefepime: RAztreonam: RImipenem/

meropenem: S

AmpCs

AmpC: GeneralChromosomal

Escherichia coliCitrobacter freundiiEnterobacter aerogenes, E. cloacaeSerratia marcescensMorganella morganiiHafnia alveiProvidencia rettgeri, P. stuartiiPseudomonas aeruginosaAeromonas sp.

AmpC: GeneralAre not inhibited by -lactamase inhibitorsNormally are repressed, so produced at low

levelsChromosomal: inducible

In all except E. coliIn the presence of certain -lactam

antibioticsNormally, produced at low levels

Plasmid-mediated also

The AmpC of E. coliChromosomal, but

not inducibleNormally expressed

at low levelsRegulated by a

growth rate-dependent attenuation mechanism

Can become highly expressed with mutations

Amp: SAmox/clav: SPiperacillin: SPip/tazo: SCefoxitin: SCeftazidime: SCeftriaxone: SCefepime: SAztreonam: SImipenem/

meropenem: S

AmpC Induction and DerepressionIs induction clinically relevant?True danger—mutation in induction

pathway“Derepressed mutant”150-1000 fold more enzyme produced than

normal

Chromosomal AmpC profileNormal

Amp: RAmox/clav: RPiperacillin: SPip/tazo: SCefoxitin: RCeftazidime: SCeftriaxone: SCefepime: SAztreonam: SImipenem/

meropenem: S

Derepressed profileAmp: RAmox/clav: RPiperacillin: RPip/tazo: RCefoxitin: RCeftazidime: RCeftriaxone: RCefepime: SAztreonam: RImipenem/

meropenem: S

Plasmid-Mediated AmpCs (pAmpC)First true proof of AmpC on plasmid: 1988

MIR-1, found in Klebsiella pneumoniae90% identical to E. cloacae ampC

Some are also inducible (DHA-1)Most frequently found in K. pneumoniaeAlso commonly found in:

K. oxytocaSalmonella sp.P. mirabilis

E. coli, E. aerogenes also

pAmpCs: DistributionWorld-wide distribution

Africa, Asia, Europe, Middle East, North America, South America, Central America

CMY-2 is most prevalent globallyAlgeria, France, Germany, Greece, India,

Pakistan, Taiwan, Turkey, UK, US

ESBLs vs AmpCs

ESBLs AmpCs

Inhibitors (pip/tazo, amp/sulbactam, amox/clav)

S R

Cefoxitin, cefotetan

S R

Ceftazidime, ceftriaxone

R R

Cefepime S/R S

Carbapenemases

CarbapenemasesCarbapenem resistance:

High level production of chromosomal AmpC with decreased outer membrane permeability (porins)E. cloacae, E. aerogenesC. freundiiE. coliS. marcescensK. pneumoniae (porins)

CarbapenemasesCarbapenem resistance:

Changes in affinity of PBPs for carbapenemsCarbapenemases

Frequently, bugs that produce a carbapenemase produce other -lactamases

CarbapenemasesKPC (plasmid, K. pneumoniae)

“Klebsiella pneumoniae carbapenemase”IMI-1 (plasmid, E. cloacae)Nmc-A (plasmid, E. cloacae)Sme-1 (plasmid S. marcescens)IMP-1 (plasmid, S. marcescens, P.

aeruginosa)L-1 (chromosomal, Stenotrophomonas

maltophilia)

Carbapenemases: ProfileR to carbapenems, penicillins,

cephalosporinsS or R to aztreonam, depending on enzymeSo the key:

Look for intermediate or R to imipenem or meropenem!

KPCInfection control emergency!!!

May test sensitive to carbapenems though!Extensive multidrug resistance (XDR)Very rapid spreadEmpiric therapy: colistin + tigecyclineKPC 1-8

Further readingYang, 2007. Ann. Pharmocother. 41:1427-

1435Jacoby, 2009. Clin. Microbiol. Rev. 22:161-

182Black et al, 2005. J. Clin. Microbiol.

43:3110-3113Livermore et al, 2001. J. Antimicrob.

Chemother. 48 Suppl 1: 87-102Pfaller and Segreti, 2006. Clin. Infect. Dis.

42: S153-163.