ESBL

DETECTION

METHODSDR.T.V.RAO MD

DR.T.V.RAO MD 1

• ESBLs are enzymes that

mediate resistance to

extended-spectrum (third

generation) cephalosporins

(e.g., ceftazidime, cefotaxime,

and ceftriaxone) and

monobactams (e.g.,

aztreonam) but do not affect

cephamycins (e.g., cefoxitin

and Cefotetan) or

carbapenems (e.g.,

meropenem or imipenem).

WHAT ARE EXTENDED-SPECTRUM

Β-LACTAMASES?

DR.T.V.RAO MD 2

3

SURVIVAL OF THE FITTEST

• Resistant bacteria survive, susceptible ones die

Mutant emerges

slowly

Sensitive cells

killed by antibiotic

Mutant’s progeny

overrun

WHY SHOULD CLINICAL LABORATORY PERSONNEL BE

CONCERNED ABOUT DETECTING THESE ENZYMES?

DR.T.V.RAO MD 4

• The presence of an ESBL-producing organism in a clinical infection

can result in treatment failure if one of the above classes of drugs is

used. ESBLs can be difficult to detect because they have different

levels of activity against various cephalosporins. Thus, the choice of

which antimicrobial agents to test is critical. For example, one enzyme

may actively hydrolyze ceftazidime, resulting in ceftazidime minimum

inhibitory concentrations (MICs) of 256 µg/ml, but have poor activity

on cefotaxime, producing MICs of only 4 µg/ml. If an ESBL is

detected, all penicillin's, cephalosporins, and aztreonam should be

reported as resistant, even if in vitro test results indicate susceptibility

SLIDE 5

DEFINITION OF ESBL

BL:

• Class A by Ambler or Group 2be by Bush classifications

• Typically, enzymes are plasmid-mediated derived from older ß-lactamases of TEM and SHV

• In early 2000s, CTX-M derived ß-lactamases are included

ΒΒΒΒ-LACTAM ANTIBIOTICS• Penicillin's

• Ampicillin

• Piperacillin

• Beta-lactam/beta-lactamase inhibitors

• Ampicillin/sulbactam

• Amoxicillin/clavulanate

• Ticarcillin/clavulanate

• Piperacillin/TazobactamDR.T.V.RAO MD 6

Β-LACTAM ANTIBIOTICS

• First Generation cephalosporins

• Cefazolin

• Cephalothin

• Second Generation oral antibiotics

• Cefuroxime (many others)

• Second Generation cephamycins

• Cefoxitin

• CefotetanDR.T.V.RAO MD 7

8

RISK FACTORS FOR ESBL INFECTION

• Length of hospital stay

• Severity of illness

• Time in the ICU

• Intubation and mechanical ventilation

• Urinary or arterial catheterization

• Previous exposure to antibiotics

Bradford PA. Clin Microbiol Rev. 2001;14:933-951.

Β-LACTAM RESISTANCE IN GRAM NEGATIVE

BACTERIA• Ampicillin resistance in Enterobacteriaceae

• Acquisition of TEM-1 β-lactamase in E. coli, SHV-1 in K. pneumonia

• Cephalosporin resistance developed by mutation of TEM and SHV

• Point mutations in TEM and SHV change structure of the enzyme

• Enables hydrolysis of cefuroxime, cephalexin, cefadroxil, cephalothin

etc…..

• Extended spectrum β-lactamases

• More TEM and SHV variants and emergence of CTX-M, VEB, PER

• Resistance to 3rd and 4th generation cephalosporins (ceftazidime,

cefotaxime)

DR.T.V.RAO MD 9

Β-LACTAM RESISTANCE IN GRAM NEGATIVE

BACTERIA

• AmpC β-lactamases

• Natural β-lactamases able to hydrolyze cephalosporins at low

level

• Mutations in regulatory genes leave to ‘derepression’ and

overexpression in Enterobacter, Serratia, Morganella spp

• Carbapenemases – resistance to cephalosporins and

carbapenems

• Acquired KPC in K. pneumoniae, Enterobacter, E.coli

• Zn dependent mettallo-enzymes (IMP, VIM) in P. aeruginosa, A.

baumanniiDR.T.V.RAO MD 10

THERE ARE MORE THAN 200 BETA-LACTAMASE TYPES IN GRAM

NEGATIVE BACILLI

• Class A: TEM-1,2; SHV-1; ESBLs, KPC

• Class B: MBLs

• Class C: AmpC

• Class D: OXA

DR.T.V.RAO MD 11

Plasmid-mediated TEM and SHV ββββ-lactamases

Ampicillin

1965

TEM-1

E.coli

S.paratyphi

1970s

TEM-1

Reported in

28 Gm(-) sp

1983

ESBL in

Europe

1988

ESBL

in USA

2000

> 130 ESBLs

Worldwide

Extended-spectrum

Cephalosporins

1963

Evolution of ββββ-Lactamases

Look and you will find ESBL

CLASSIFICATION OF Β LACTAMASES

• Richards and Sykes (1971)

• substrate

• Ambler (1969)

• structure

• Bush, Jacoby, Medeiros (1995)

• Substrate; correlation with molecular structure

• 150 TEM;

• 88 SHV;

• 88 OXA,

• 53 CTX-M;

• 22 IMP;

• 12 VIM + smaller number of other enzymes (http://www.lahey.o

Plasmid-mediated TEM and SHV ββββ-lactamases

Ampicilli

n

1965

TEM-1

E.coli

S.paratyphi

1970s

TEM-1

Reported in

28 Gm(-) sp

1983

ESBL in

Europe

1988

ESBL

in USA

2000

> 130 ESBLs

Worldwide

Extended-spectrum

Cephalosporins

1963

Evolution of ββββ-Lactamases

14DR.T.V.RAO MD 14

15

AMBLER CLASSIFICATION OF Β-LACTAMASES

Active site

Nucleotide

sequence

Four evolutionarily distinct molecular classes

A C D

Serine-enzymes

B

Zinc-enzymes

β-lactamases

DR.T.V.RAO MD

16

MODIFIED BUSH–JACOBY–MEDEIROS

CCLASSIFICATION OF Β–LACTAMASES

Functional Substrate profile Group

Molecular Class

Inhibitor

Example

1 Cephalosporinase C Oxa AmpC, MIR-1

2a Penicillinase A Clav. S.aureus

2b Broad spectrum A Clav. TEM-1/2, SHV-1

2be Extended spectrum A Clav. TEM 3-29, TEM46-104 SHV2-28, CTX-M types

2br Inhibition resistant A - TEM 30-41 (IRT1-12)

2c Carbenicillinase A Clav. PSE-1

2d Oxacillinase D (Clav.) OXA-1 (OXA-2 &-10 derived ESBL)

2e Cephalosporinase A Clav. FPM-1 P. vulgaris, CepA B. fragilis.

2f Carbapenemase A Clav. IMI-1, NmcA, Sme 1-3

3 Metallo-enzyme B - S.maltophilia

4 Penicillinase - - B.cepacia

DR.T.V.RAO MD

AMPC ΒΒΒΒ-LACTAMASES

• Chromosomally encoded-cell wall turnover

• Enterobacter sp., Citrobacter sp., Serratia sp., Morganella sp. Even E. coli.

• Third-generation cephalosporins are not good inducers of AmpC β-lactamase

• Third-generation cephalosporin resistant strains are derepressed—meaning that the AmpC β-lactamase is not inducible anymore.

• AmpC mutants are cephamycin resistant

DR.T.V.RAO MD 17

AMPC ββββ-LACTAMASES

• Molecular class C, functional group 1

• Not inhibited by CA

• Confers resistance to penicillins, cephalosporins, monobactam, and cephamycin

• Chromosomally- or plasmid-mediated

• Many genera in Enterobacteriaceae encode chromosomal inducible AmpC

• Serratia marcescens

• Enterobacter cloacae

• Citrobacter freundii

• Morganella morganii

• Hafnia alvei

• Yersenia enterocolitica

• Pseudomonas aeruginosa

AMPC ββββ-LACTAMASES

• Expression of the chromosomal ampC is generally low

• Inducible in response to certain ββββ-lactams

• Factors involved in ampC induction:

• ββββ-lactam interaction with PBPs

• Byproducts of cell wall synthesis

• Gene products

• AmpR

• AmpD

• AmpG

20

CTX-M• Fast growing – important group

• Preferentially hydrolyse, and confer resistance to cefotaxime

• Escape of chromosomal ß-lactamase genes from Kluyvera spp (a bug

of no clinical importance!)

• Having migrated to mobile DNA, CTX-M ß-lactamases genes may

evolve further – undergoing mutations that increase activity against

ceftazidime

• The first CTX-M ESBL in the UK was found as recently as 2000, in a

solitary isolate of K. oxytoca

• First outbreak, caused by K. pneumoniae producing the new enzyme

CTX-M-26, was recorded in Birmingham in 2001Livermore D and Hawkey P. J Antimicrob Chemother 2005; 56: 451-454

HPA Report September 2005 www.hpa.org.uk/publications

21

CTX-M• Has supplanted TEM and SHV types as the predominant

ESBLs in the UK

• CTX-M-15 enzyme most common in UK

• 28/105 cases resulted in death in one UK PCT

• Most CTX-M-15 producing E. Coli isolates tested by HPA were multi-resistant to aminoglycosides, fluoroquinolones and trimethoprim as well as all ß-lactams, except carbapenems and temocillin

HPA Report September 2005 www.hpa.org.uk/publications

22

INCREASING NUMBERS OF ESBLS

0

10

20

30

40

50

60

70

80

2000 2001 2002 2003 2004 2005 2006 2007

# of ESBLs per year

Lewis, et al. AAC 51:4015, 2007

23

BETA-LACTAMASE INHIBITORS

• Resemble β-lactam antibiotic structure

• Bind to β-lactamase and protect the antibiotic from destruction

• Most successful when they bind the β-lactamase irreversibly

• Three important in medicine

• Clavulanic acid

• Sulbactam

• Tazobactam

DR.T.V.RAO MD

24

TYPES OF ESBLS

• TEM

• SHV

• CTX-M

• OXA

Mutations

ESBL PhenotypePlasmid-mediated

DR.T.V.RAO MD

ESBLS ARE BETA-LACTAMASES WHICH:

• Hydrolyse third generation cephalosporins

(and aztreonam, penicillins and many other

cephalosporins)

• Do not appreciably hydrolyse cephamycins

(cefoxitin or Cefotetan) or carbapenems

• Are inhibited by beta-lactamase inhibitors

such as clavulanic acid

DR.T.V.RAO MD 25

SLIDE 26

HISTORICAL PERSPECTIVES

LABORATORY DETECTION (V-1)1988

Jarlier effect – CTX with Augmentin (Jarlier V et al Rev Infect Dis 1988)

1990

NCCLS– ceftazidime zone <15mm Kirby Bauer Method for screening

1994

Synergy testing with ceftazidime (Sader HS et al Diagn Microbiol Infect Dis 1994)

LABORATORY DETECTION OF ESBL

• Phenotypic Methods

•Screening methods

•Confirmatory Methods

• Genotypic Methods

Why Test for β-lactamases ?

� Improve clinical outcome

� Inappropriate treatment leads to poor outcome

� Each 1 hour delay increases mortality by 7.6% in septic shock1

� Encourage antimicrobial stewardship

� Spare carbapenems..

� Reduce C. difficile / antibiotic associated diarhoea

� Enhanced surveillance

� Identify emerging resistance problems

� Develop structures to prevent dissemination

� Infection Control

� ‘Search and Destroy’ analogous to MRSA ?

� Laboratory Detection is not always easy… OR Rapid

1Kumar, Crit Care Med, 2006

SLIDE 29

LABORATORY DETECTION

1996

Etest with ceftazidime and clavulanate was recommended (Cormican MG et al JCM)

1996

>50% ESBL E. coli and 29% of ESBL K. pneumoniae were resistant to cefoxitin and 10% of non-ESBL E.coli and K. pneumoniae also resistant to cefoxitin Jacoby GA & Han P JCM )

2001

Cefpodoxime recommended for screening Clin Microbiol Rev 2001

WHY DETECT ESBL PRODUCERS?

• ESBL producers may:

• Appear Sensitive to some cephalosporins s in vitro

• Show major inoculum effects

• Fail in therapy, despite appearing susceptible

DR.T.V.RAO MD 30

ESBL PRODUCERS FREQUENTLY

APPEAR SUSCEPTIBLE TO

CEPHALOSPORINS

0

10

20

30

40

50

60

70

80

<=8

(S)

>=32

(R)

Cefotax.

Ceftriax.

Ceftaz.

• Enterobacteriaceae are

traditionally reported as

susceptible to ceftazidime,

cefotaxime, ceftriaxone,

aztreonam, and cefepime

when MIC <= 8 µg/mL

32

CHOICE OF INDICATOR CEPHALOSPORIN

• TEM & SHV – obvious resistance to ceftazidime, variable to

cefotaxime

• CTX-M – obvious resistance to cefotaxime, variable to

ceftazidime

• All ESBLs – obvious resistance to cefpodoxime

• Cefuroxime, cephalexin and cephradine are unreliable

indicators

Livermore D and Woodford N HPA Guidance 2004

SLIDE 33

CURRENT MODERN METHODS

• CLSI – Clinical Laboratory and Standards Institute

• ARMRL - Antibiotic Resistance Monitoring and Reference

Laboratory, Health Protection Agency Centre

for Infections, London

• EUCAST- European Society of Clinical Microbiology &

Infectious Diseases

• Commercial methods – Etest, BD Phoenix, Vitek, Neo-

tabs & others

• Klebsiella pneumoniae

• Escherichia coli

• Proteus mirabilis

• Enterobacter cloacae

• Non-typhoidal Salmonella

(in some countries)

COMMON ESBL PRODUCERS

DR.T.V.RAO MD 34

DIVERSITY OF ESBL’S

• Confer resistance to 1st , 2nd, 3rd cef.

• Most are susceptible to β-lactamase inhibitors

• Most are susceptible to 4th cef.

• All are susceptible to carbapenems

• Diversity of ESBL

• SHV (widespread)

• TEM (>100 types)

• OXA

• Predominantly in Pseudomonas

• less susceptible to β-lactamase inhibitors

• CTX-M

• Probably independent evolution

• Highly resistant to 3rd generation cephalosporines

• initially in South America, Far East & Eastern Europe

• Probably most frequent worldwide

DETECTION STRATEGY: STEP 1

• Screen Enterobacteriaceae with :

• Cefpodoxime- best general ESBL substrate

• Cefotaxime & ceftazidime- good substrates

for CTX-M & TEM/SHV, respectively

DR.T.V.RAO MD 36

DETECTION OF ESBLS: STEP 2

• Seek ceph/clav synergy in ceph R isolates

•Double disc

•Combination disc

•Etest

DR.T.V.RAO MD 37

COMBINATION DISK METHODCARTER MW ET AL: J CLIN MICROBIOL 2000; 38: 4228 -

4232

Difference > 5 mm

ESBL CONFIRMATORY TESTS

Double-disk synergy (DDS) test

• CAZ and CAZ/CA disks

• CTX and CTX\CA disks

• Confirmatory testing

requires using both CAZ

and CTX alone and with CA

• 5 mm enhancement of the inhibition

zone of antibiotic/CA combination vs antibiotic

tested alone = ESBL

DR.T.V.RAO MD 40

DR.T.V.RAO MD 41

42

ESBLS DETECTION METHODS:

INHIBITION BY CLAVULANIC ACID

Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and

aztreonam discs (30 mcg each)

ESBL DETECTION : COMBINATION DISCS: +VE RESULT, ZONE ENLARGED 50%

Discs (30+10 µµµµg) % Detected (n =100)

Ceftazidime +/- clav 88

Cefotaxime +/- clav 66

Both 93

M’Zali et al. 2000, JAC, 45, 881

DR.T.V.RAO MD 43

ESBL DETECTION

•2 Steps:

– Screen cefpodoxime ; cefotaxime & ceftazidime

– Synergy test with ceph/clav

•Combination discs are most cost effective synergy tests; Etests a good alternative.. or automate

•Guidelines on http//www.hpa.org.uk- type ESBL in search facility

DR.T.V.RAO MD 44

COMPARING DISK DIFFUSION WITH MINIMUM

INHIBITORY CONCENTRATIONS

Disk diffusion MICs

cefpodoxime < 22 mm cefpodoxime > 2 µg/ml

ceftazidime < 22 mm ceftazidime > 2 µg/ml

aztreonam < 27 mm aztreonam > 2 µg/ml

cefotaxime < 27 mm cefotaxime > 2 µg/mlDR.T.V.RAO MD 45

Etest for ESBLs

Cefotaxime

Cefotaxime

+

clavulanateDR.T.V.RAO MD 46

ESBL Confirmatory Test

Positive for ESBL

Ceftaz/CACefotax/CA

Ceftaz Cefotax

47DR.T.V.RAO MD 47

48

ESBL CONFIRMATORY TEST NEGATIVE FOR

ESBL

Ceftaz/CA Cefotaxime/CA

Ceftaz Cefotax

48DR.T.V.RAO MD

ESBL CONFIRMATORY TEST

Ceftaz/CA CeftazEtest

49DR.T.V.RAO MD 49

PITFALLS IN ESBL DETECTION

• Methods optimised for E. coli & Klebsiella

• More difficult with Enterobacter

– clavulanate induces AmpC; hides ESBL

• Best advice is to do synergy test (NOT SCREEN) with 4th

gen ceph

DR.T.V.RAO MD 50

SYNERGY TESTS WITH 4-GEN CEPHS

•Cefepime/clav (Mast & AB Biodisk)

•Cefpirome clav (Oxoid)

• Devt. driven by spread of clonal E. aerogenes with

TEM-24 in Belgium & France

• Sensitivity for weak ESBLs remains to be proven

• Cefpirome & cefepime products need comparison

DR.T.V.RAO MD 51

CEPH R BUT SYNERGY –VE…

AmpC- plasmid or

chromosomal

S to 4 gen cephs

K1 hyperproducer

K. oxytoca

R cefuroxime, aztreonam, cefpodoxime

S ceftazidime, I to cefotaxime

May give false +ve ESBL test

Impermeable E.

coli, Kleb

R cefoxitin & cefuroxime; not ¾-gen cephs

Carbapenemase

Metallo or not

R includes imipenem & / or meropenem

DR.T.V.RAO MD 52

BACTERIA NOT TO TEST FOR ESBLS

•Acinetobacter

– Often S to clavulanate alone

•S. maltophilia

– +vet result by inhibition of L-2 chromosomal β-

lactamase, ubiquitous in the species

DR.T.V.RAO MD 53

ESBL REPORTING RULE

• The rule (CLSI =NCCLS) M100-S15)H• “Strains of Klebsiella spp. E. coli, and Proteus mirabilis that produce

ESBLs may be clinically resistant to therapy with penicillin's,

cephalosporins, or aztreonam, despite apparent in vitro susceptibility

to some of these agents.”

• The messageH• Report “confirmed” ESBL-producing strains as R to all penicillin's,

cephalosporins, and aztreonam

54DR.T.V.RAO MD 54

WILL CLSI CONFIRMATORY TEST DETECT ALL

ESBL-PRODUCING GNR?

• No - some isolates have ESBLs plus other resistance mechanismsthat mask ESBL detection in the confirmatory test, e.g.,

• > 1 ESBL

• ESBL + AmpC

• ESBL + porin mutation

• ESBLs occur in species other than E. coli, Klebsiella spp., and Proteus mirabilis which CLSI does not currently address

55

• PCR and sequencing

• The gold standard

• Can detect all variants

• Easy to perform

• Labor intensive

MOLECULAR DETECTION OF ESBLS

• 144 putative of ESBL producers

• ESBL detection:

• AS: Microscan, Vitek2, Phoenix

• Phenotypic tests: Etest, DDS

• Molecular tests: PCR, IsoElectric Focusing (IEF)

• Molecular identification: the reference method

• JCM. Apr. 2007, p.1167-1174

ESBL DETECTION: AUTOMATED

SYSTEMS (AS)

ESBL DETECTION: AUTOMATED

SYSTEMS

Detection

Method

Sensitivity

%

Specificity

%

PPV

%

NPV

%

MicroScan 83.5 72.9 81.6 75.4

Phoenix 98.8 52.2 75 96.6

Vitek2 85.9 78 84.9 79.3

DDS 92.9 96.6 97.5 90.5

Etest 94.1 84.7 89.9 90.9

JCM. Apr. 2007, p.1167-1174

PROBLEMATIC ORGANISMS….• ESBLA organisms with AmpC (Enterobacter, Citrobacter, Serratia)

• AmpC is induced by calvulanate

• Use cefipime in synergy tests

• ESBLCARBA

• Mettallocarbapenemases (Pseudomonas, Acinetobacter)

• Synergy with EDTA

• Hodge test

• ESBLM

• Difficult !

• Boronic acid for plasmidic AmpC

• Numerous commercial disc systems

• AmpC and ESBL inhibitors

60

MICROBIOLOGY LABORATORIES

AND ESBLS

• Unfortunately ,many clinical laboratories lack of understanding regarding ESBLs and Ampc ß-lactamase and their detection .This has been documented in a study in Connecticut USA, where it was found that 21% of laboratories failed to detect extended –spectrum cephalosporins and Aztreonam in ESBLs and Ampc.

• The true prevalence of ESBLs is not known and is probably underestimated because of difficulties encounter in their detection. However ,it is clear that ESBLs –producing organisms are distributed worldwide and their prevalence is increasing.

CARBAPENEMS - TREATMENT OF CHOICE FOR

SERIOUS INFECTIONS WITH ESBL PRODUCERS

• Carbapenems are not hydrolyzed by ESBLs to any great

extent

• Success rates with carbapenems for ESBL producers

consistently exceed 80%, and in no study has the outcome

with carbapenems been surpassed [Paterson CID 2004; Bhavnani DMID 2006; Zanetti AAC 2003]

HAND WASHING STILL CAN REDUCE THE ESBL

SPREAD

DR.T.V.RAO MD 62

DR.T.V.RAO MD 63

Created by Dr.T.V.Rao MD for ‘ ‘e-learning’ resources

on implication of misuse of Antibiotics and

consequences for Medical and Paramedical students in

Developing World

Email

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