ESBL

DETECTION
METHODS
DR.T.V.RAO MD

DR.T.V.RAO MD 1
 WHAT ARE EXTENDED-SPECTRUM
Β-LACTAMASES?
• 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).

DR.T.V.RAO MD 2
 SURVIVAL OF THE FITTEST
• Resistant bacteria survive, susceptible ones die

Mutant emerges Sensitive cells Mutant’s progeny

slowly killed by antibiotic overrun
3
 WHY SHOULD CLINICAL LABORATORY PERSONNEL BE
CONCERNED ABOUT DETECTING THESE ENZYMES?
• 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

DR.T.V.RAO MD 4
 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
SLIDE 5
 Β-LACTAM ANTIBIOTICS
• Penicillin's
• Ampicillin
• Piperacillin
• Beta-lactam/beta-lactamase inhibitors
• Ampicillin/sulbactam
• Amoxicillin/clavulanate
• Ticarcillin/clavulanate
• Piperacillin/Tazobactam
DR.T.V.RAO MD 6
 Β-LACTAM ANTIBIOTICS
• First Generation cephalosporins
• Cefazolin
• Cephalothin
• Second Generation oral antibiotics
• Cefuroxime (many others)
• Second Generation cephamycins
• Cefoxitin
• Cefotetan
DR.T.V.RAO MD 7
 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

8
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.
baumannii
DR.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
Evolution of β-Lactamases
Plasmid-mediated TEM and SHV β-lactamases

Extended-spectrum
Ampicillin
Cephalosporins
1963 1965 1970s 1983 1988 2000

Look and you will find ESBL

TEM-1 TEM-1 ESBL in ESBL > 130 ESBLs

E.coli Reported in Europe in USA Worldwide
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;
• 12 VIM + smaller number of other enzymes (http://www.lahey.o
 Evolution of β-Lactamases
Plasmid-mediated TEM and SHV β-lactamases

Ampicilli Extended-spectrum
n Cephalosporins
1965 1970s 1983 1988 2000

TEM-1 TEM-1 ESBL in ESBL > 130 ESBLs

1963 E.coli Reported in Europe in USA Worldwide
S.paratyphi 28 Gm(-) sp

DR.T.V.RAO MD 14
14
 AMBLER CLASSIFICATION OF Β-
LACTAMASES

β-lactamases

Active site Serine-enzymes Zinc-enzymes

Nucleotide
sequence A C D B

Four evolutionarily distinct molecular classes

DR.T.V.RAO MD 15
 MODIFIED BUSH–JACOBY–MEDEIROS
CCLASSIFICATION OF Β–LACTAMASES

Functional Substrate profile Molecular Inhibitor Example

Group Class

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
DR.T.V.RAO MD 16
4 Penicillinase - - B.cepacia
 AMPC Β-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 β-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 β-LACTAMASES
• Expression of the chromosomal ampC is
generally low
• Inducible in response to certain β-lactams
• Factors involved in ampC induction:
• β-lactam interaction with PBPs
• Byproducts of cell wall synthesis
• Gene products
• AmpR
• AmpD
• AmpG
 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 2001
Livermore D and Hawkey P. J Antimicrob Chemother
20 2005; 56: 451-454
HPA Report September 2005 www.hpa.org.uk/publications
 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

21
HPA Report September 2005 www.hpa.org.uk/publications
 INCREASING NUMBERS OF ESBLS
80
70
# of ESBLs per year

60
50
40
30
20
10
0
2000 2001 2002 2003 2004 2005 2006 2007

Lewis, et al. AAC 51:4015, 2007 22

 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 23
 TYPES OF ESBLS
• TEM
• SHV
• CTX-M Mutations
• OXA

ESBL Phenotype
Plasmid-mediated
DR.T.V.RAO MD 24
 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
 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)

SLIDE 26
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

 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

SLIDE 29
 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
80
• Enterobacteriaceae are
70
traditionally reported as
60
susceptible to ceftazidime,
50
Cefotax.
cefotaxime, ceftriaxone,
40
Ceftriax. aztreonam, and cefepime
30
Ceftaz. when MIC <= 8 µg/mL
20
10
0
<=8 >=32
(S) (R)
 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

32
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
• EUCAST- European Society of Clinical Microbiology &
Infectious Diseases
• Commercial methods – Etest, BD Phoenix, Vitek, Neo-
tabs & others

SLIDE 33
 COMMON ESBL PRODUCERS
• Klebsiella pneumoniae
• Escherichia coli
• Proteus mirabilis
• Enterobacter cloacae
• Non-typhoidal Salmonella
(in some countries)

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 METHOD
CARTER 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
 ESBLS DETECTION METHODS:
INHIBITION BY CLAVULANIC ACID

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

aztreonam discs (30 mcg each)

42
 ESBL DETECTION : COMBINATION DISCS: +VE RESULT,
ZONE ENLARGED 50%

D iscs (30+10 µ g) % D etected (n =100)

C eftazidim e +/- clav 88

C efotaxim e +/- clav 66

B oth 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/ml

DR.T.V.RAO MD 45
 Etest for ESBLs
Cefotaxime

Cefotaxime
+
clavulanate
DR.T.V.RAO MD 46
 ESBL Confirmatory Test
Positive for ESBL
Cefotax/CA
Ceftaz/CA

Ceftaz Cefotax

DR.T.V.RAO MD 47
47
 ESBL CONFIRMATORY TEST NEGATIVE FOR
ESBL

Ceftaz/CA Cefotaxime/CA

Ceftaz Cefotax

DR.T.V.RAO MD 48
48
 ESBL CONFIRMATORY TEST
Etest Ceftaz/CA Ceftaz

DR.T.V.RAO MD 49
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 S to 4 gen cephs
chromosomal

K1 hyperproducer R cefuroxime, aztreonam, cefpodoxime

K. oxytoca
S ceftazidime, I to cefotaxime
May give false +ve ESBL test

Impermeable E. R cefoxitin & cefuroxime; not ¾-gen cephs

coli, Kleb

Carbapenemase R includes imipenem & / or meropenem

Metallo or not
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

DR.T.V.RAO MD 54
54
 WILL CLSI CONFIRMATORY TEST DETECT ALL
ESBL-PRODUCING GNR?
• 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
• ESBLs occur in species other than E. coli, Klebsiella spp., and
Proteus mirabilis which CLSI does not currently address

55
 MOLECULAR DETECTION OF ESBLS
• PCR and sequencing
• The gold standard
• Can detect all variants
• Easy to perform
• Labor intensive
 ESBL DETECTION: AUTOMATED
SYSTEMS (AS)
• 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
Detection Sensitivity Specificity PPV NPV
Method % % % %
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
 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.

60
 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
 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
doctortvrao@gmail.com

DR.T.V.RAO MD 63