ESBL
EXTENDED SPECTRUM BETA LACTAMASES
DYNAMICS AND DETECTION
Dr.T.V.Rao MD
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DR.T.V.RAO
SURVIVAL OF THE FITTEST
Resistant bacteria survive, susceptible
ones die
Mutant emerges
slowly
2
Sensitive cells
killed by antibiotic
Mutant’s progeny
overrun
PENICILLIN
BETA LACTAM RING
BETA LACTAMASES enzymes that
inactivate the beta-lactam ring
CEPHALOSPORIN
Dr.T.V.Rao MD
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BETA LACTAM RING
ACTION OF A B-LACTAMASE
S
H2O
N
O
Inactive penicilloate
COOH
S
Active penicillin
HN
Dr.T.V.Rao MD
OH
COOH
4
O
BASIS OF BETALACTAMSE
ACTIVITY
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Time-delayed Growth. Betalactamase (red) is
produced by the central
colony, promoting growth
of nearby, non-resistant
colonies as it deactivates
ampicillin (blue). Diffusion
of beta lactamase through
agar leads to time-delayed
growth of non-resistant
colonies
B-LACTAM ANTIBIOTICS
Penicillin's
•Ampicillin
•Piperacillin
Beta-lactam/beta-lactamase inhibitors
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•Ampicillin/sulbactam
•Amoxicillin/clavulanate
•Ticarcillin/clavulanate
•Piperacillin/Tazobactam
PENICILLINS
1st gen – strep infection (G+) Ex. Penicillin,
Cloxacillin
Extended-spectrum – have broader
spectrum against G- including E.Coli Ex.
Amoxicillin With inhibitor would protect
against some beta-lactamase producers Ex.
Amoxicillin/Clav
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Broad spectrum – many
Enterobacteriaceae Ex
Piperacillin/Tazobactam
Some beta-lactamases only inactivate a
small number of antibiotics e.g. penicillin
extended
spectrum to all the penicillins and
Others have
cephalosporins e.g. cefuroxime,
ceftriaxone (ESBLs)
Dr.T.V.Rao MD
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In addition may also carry resistance to
other antibiotics e.g. ciprofloxacin.
DEFINITION OF ESBL
:
Dr.T.V.Rao MD
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Class A by Ambler or Group 2be
by Bush classifications
Typically, enzymes are plasmidmediated derived from older ßlactamases of TEM and SHV
In early 2000s, CTX-M derived ßlactamases are included
ESBL EVOLUTION
Mid 1980s
Variants of TEM and SHV
Breakdown 3rd generation
cephalosporins
Mainly in hospital Klebsiella
Dr.T.V.Rao MD
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Spread world wide
WHAT ARE EXTENDED-SPECTRUM
Β-LACTAMASES?
Dr.T.V.Rao MD
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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).
AMBLER CLASSIFICATION OF ΒLACTAMASES
β-lactamases
Active site
Nucleotide
sequence
Serine-enzymes
Zinc-enzymes
C
B
A
D
Dr.T.V.Rao MD
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Four evolutionarily distinct molecular classes
WHAT IS A BETA-LACTAM?
Abx
• Penicillin
• Cephalosporin
• Monobactam
• Carbapenem
Bacteriocidal
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Google Images
CEPHALOSPORINS
4th
3rd
2nd
Willey, et al., 2008
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1st
CEPHALOSPORINS-USES
1st gen: strep, staph, G- including E.coli Ex.
Cefazolin
2nd gen: greater spectrum against G- Ex.
Cefoxitin
3rd gen: even greater activity, combat
narrow-spectrum beta-lactamase producers
 ESBLs emerged Ex. Ceftazidime
Dr.T.V.Rao MD
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4th gen: effective against G- bacilli
expressing Xm AmpC resistant to 3rd gen
Ex. Cefepime
OTHERS
Monobactams
Monobactams very active against Gincluding E.coli Ex. Aztreonam
Carbapenems
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Carbapenems have an extremely
broad spectrum. Cross-reactivity with
penicillins or cephalosporins Ex.
Imipenam
THE FIGHT GOES ON ..
Beta-lactam
Beta-lactamase
Beta-lactamase inhibitor
Google Images
Dr.T.V.Rao MD
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ESBL
COMMON ESBL PRODUCERS
Klebsiella
pneumoniae
Escherichia coli
Proteus mirabilis
Enterobacter cloacae
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Non-typhoidal
Salmonella (in some
countries)
THE FIGHT
BETA-LACTAM
PG
Dr.T.V.Rao MD
cell
LYSIS
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O
N
THE FIGHT
BETA-LACTAMASE
PG
beta-lactamase
Dr.T.V.Rao MD
cell
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O
N
THE FIGHT
BETA-LACTAMASE
PG
O
NH
OH
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cell
THE FIGHT
BETA-LACTAMASE INHIBITOR
PG
beta-lactamase
Dr.T.V.Rao MD
Inhibitor
cell
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O
N
THE FIGHT
BETA-LACTAMASE INHIBITOR
PG
beta-lactamase
Inhibitor
Dr.T.V.Rao MD
cell
LYSIS
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O
N
ESBLS
Enterobacteriaceae
Resistance to oxyimino-cephalosporins and Monobactams
but not cephamycins and carbapenem
• Susceptible to beta-lactamase inhibitors
•Genes
SHV
TEM
CTX-M
OXA
Oteo, et al., 2010
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AmpC
Evolution of -Lactamases
Plasmid-mediated TEM and SHV -lactamases
Ampicillin
1963
1965
Extended-spectrum
Cephalosporins
1970s
1983
1988
2000
Look and you will find ESBL
D
r
.
ESBL in
Europe
ESBL
in USA
> 130 ESBLs
Worldwide
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TEM-1
TEM-1
E.coli
Reported in
S.paratyphi 28 Gm(-) sp
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;
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• 12 VIM + smaller number of other enzymes (http://www.lahey.o
CLASSIFICATION
Ambler Classification
• Molecular class A – D
•A
Bush-Jacoby-Medeiros Classification
• Functional group 1 – 4
Paterson and Bonomo, 2005
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•2
• 2b
• 2be
BETA-LACTAMASE
INHIBITORS
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Resemble β-lactam antibiotic structure
Bind to β-lactamase and protect the
antibiotic from destruction
Most successful when they bind the βlactamase irreversibly
Three important in medicine
•Clavulanic acid
• Sulbactam
• Tazobactam
TEM
ESBL
CTX-M
K1
Ceftazidime
AmpC
Hi-level
R
R
v
S
Cefotaxime
R
v
R
S
Cefoxitin
R
S
S
S
Aztreonam
R
v
v
R
Synergy + clav
No
+++
+++
No
Dr.T.V.Rao MD
Know the species
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RESISTANCE AND GENETICS
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
TYPES OF ESBLS
TEM
SHV
CTX-M
Mutations
OXA
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Plasmid-mediated
ESBL Phenotype
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
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cephradine
are unreliable indicators
Livermore D and Woodford N HPA Guidance 2004
CURRENT MODERN METHODS
CLSI – Clinical Laboratory and Standards
Institute
ARMRL - Antibiotic Resistance Monitoring
and Reference Laboratory, Health
Protection Agency Centre for Infections,
London
Commercial methods – Etest, BD Phoenix,
Vitek Neo tabs & others
Slide
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EUCAST- European Society of Clinical
Microbiology & Infectious Diseases
DETECTION OF ESBLS
Seek ceph/clav synergy in ceph R
isolates
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•Double disc
•Combination disc
•Etest
CHALLENGES FOR THE
DIAGNOSTIC LAB
Detection…. Hemophilus, Neisseria etc.
Predicting -lactamase types. Have GNB got ?:
 ESBL,
AmpC
Metallo types, VIM, IMP etc…
Spotting unusual patterns; knowing what to refer ???
DETECTION STRATEGY: STEP 1
Screen Enterobacteriaceae
with :
• Cefpodoxime- best general
ESBL substrate
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• Cefotaxime & ceftazidime- good
substrates for CTX-M &
TEM/SHV, respectively
COMBINATION DISK METHOD
CARTER MW ET AL: J CLIN MICROBIOL 2000; 38: 4228 - 4232
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Difference > 5 mm
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Dr.T.V.Rao MD
KLEBSIELLA PNEUMONIA PRODUCING A HIGHER
The higher level of
ESBL production is
indicated by the
inhibition of the
β-lactamase by
clavulanic acid and
the resulting elliptical
inhibitory zone
between cefotaxime
(CTX 5) and
Augmentin (AMC 60).
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ACTIVITY ESBL
Double disc antagonism for inducible AmpC
Dr.T.V.Rao MD
Ceftazidime
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Cefoxitin
AMPC INDUCIBILITY- WHEN TO LOOK
Rarely!!!!!
Risk is mutation, not inducibility per se
Best to identify & predict risk from species
Biggest risk Enterobacter & C freundii
Avoid cephalosporins against them
Identify means identify TO SPECIES LEVEL all
Enterobacteriaceae (‘coliforms’) ex serious infections
ESBLS DETECTION METHODS:
INHIBITION BY CLAVULANIC ACID
Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and
aztreonam discs (30 mcg each)
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ESBL DETECTION
Dr.T.V.Rao MD
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– Screen cefpodoxime
; cefotaxime &
ceftazidime
– Synergy test with
ceph/clav
Combination discs are
most cost effective
synergy tests; Etests
a good alternative.. or
automate
ESBL Confirmatory Test
Positive for ESBL
Ceftaz
Dr.T.V.Rao MD
Cefotax
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Ceftaz/CA
Cefotax/CA
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ESBL CONFIRMATORY TEST
NEGATIVE FOR ESBL
Ceftaz
Dr.T.V.Rao MD
Cefotaxime/CA
Cefotax
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Ceftaz/CA
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ESBL CONFIRMATORY TEST
Dr.T.V.Rao MD
Ceftaz
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Etest Ceftaz/CA
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ESBLS: TIMES A’ CHANGING WITH CTX-M
Old advice- test ceftazidime; ESBL test if R
New advice- test ceftazidime & cefotaxime;
ESBL test if R to either
• Still true- Only testing cefuroxime is
inadequate
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• Alternative- test cefpodoxime; ESBL
test if R
COMPARING DISK DIFFUSION WITH MINIMUM
INHIBITORY CONCENTRATIONS
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/ml
ceftriaxone < 25 mm
ceftriaxone > 2 µg/ml
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Disk diffusion
ESBL CONFIRMATORY TESTS
• 5 mm enhancement of the inhibition
zone of antibiotic/CA combination vs antibiotic
tested alone = ESBL
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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
SYNERGY TESTS WITH
4-GEN CEPHALOSPORINS
Cefepime/clav (Mast & AB Biodisk)
Cefpirome clav (Oxoid)
• Devt. driven by spread of clonal E.
aerogenes with TEM-24 in Belgium &
France
• Cefpirome & cefepime products need
comparison
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• Sensitivity for weak ESBLs remains to
be proven
PITFALLS IN ESBL DETECTION
•Methods optimised for E. coli &
Klebsiella
•More difficult with Enterobacter
– clavulanate induces AmpC; hides ESBL
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•Best advice is to do synergy test (NOT
SCREEN) with 4th gen ceph
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
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Dr.T.V.Rao MD
BACTERIA NOT TO TEST FOR
ESBL’S
Acinetobacter
– Often S to
clavulanate alone
S. maltophilia
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– +vet result by
inhibition of L-2
chromosomal lactamase,
ubiquitous in the
species
ESBL REPORTING RULE
The rule (CLSI =NCCLS) M100-S15)…
• “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 message…
• Report “confirmed” ESBL-producing strains as R to all
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penicillin's, cephalosporins, and aztreonam
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WILL CLSI CONFIRMATORY TEST
DETECT ALL ESBL-PRODUCING GNB?
No - some isolates have ESBLs plus other
resistance mechanisms that mask ESBL
detection in the confirmatory test, e.g.,
• > 1 ESBL
• ESBL + AmpC
• ESBL + porin mutation
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ESBLs occur in species other than E. coli,
Klebsiella spp., and Proteus mirabilis which
CLSI does not currently address
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ESBL DETECTION:
AUTOMATED SYSTEMS (AS)
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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
THE RESISTANCE BECOMING
COMPLEX
Beta-lactamases are getting more complex
Full I/D needs complex molecular methods
Much can be inferred from simple tests.
Needs I/D
Knowledge of what’s unusual
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Testing wide panels of antibiotics; synergy
tests
ANTIBIOTIC POLICY CHANGES PRACTISED
Nitrofurantion substituted for quinolones
in UTIs
Imipenem substituted for quinolones in
serious sepsis
Ertapenem introduced for ESBL sepsis
Gentamicin substituted for
cephalosporins in surgical prophylaxis
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Return to amoxycillin in respiratory tract
infections
MICROBIOLOGY LABORATORIES
AND ESBL’S
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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.
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HAND WASHING STILL CAN REDUCE
THE ESBL SPREAD
The programme created by Dr.T.V.Rao MD
for basic understanding by Medical
Microbiologists in the Developing World
Email
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doctortvrao@gmail.com