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Introduction to

L
or re
β-lactamases

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L
Irene Galani, PhD

by e
Infectious Diseases Laboratory,

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Molecular Biology Section,
n
4th Department of Internal Medicine,
O
Athens University School of Medicine
ID

egalani@med.uoa.gr
M
C
ES
 ry
β-lactamases

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►Beta-lactamases are bacterial enzymes that hydrolyze the beta-

or re
lactam ring and render the antibiotic inactive before it reaches the

th tu
PBP target.

au ec
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by e
© lin
n
O
ID
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C
ES
 ry
Sir Alexander Fleming Discovers Penicillin

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In 1928

or re
th tu
fA mold on a petri dish was

au ec
observed to inhibit growth of

L
Staphylococcus bacteria.

by e
© lin
f The active ingredient
n isolated from this mold was
O
found to be a safe and
ID

effective bacteria-killing
M

agent of enormous potency.

C
ES
 f Fleming published his discovery in 1929, in the

ry
British Journal of Experimental Pathology, but

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little attention was paid to his article.

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n
O
ID
M

f Use of penicillin did not began until

C

the 1940s when Howard Florey and

ES

Ernst Chain isolated the active

ingredient and developed a powdery
form of the medicine.
ES
C
M
ID
O
n
© lin
by e
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au ec
The first β-lactamase

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ry
ES
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ID
O
n
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by e
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au ec
S. aureus penicillinase

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ry
 ry
1945 Nobel Prize Acceptance Speech

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Sir Alexander Fleming warns

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of the danger of resistance:

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au ec
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“It is not difficult to make

by e
microbes resistant to penicillin

© lin
in the laboratory by exposing
them to concentrations not
n
O
sufficient to kill them, and the
Fleming (centre) receiving the Nobel prize same thing has occasionally
ID

from King Gustaf V of Sweden (right), happened in the body…”

1945
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ES
 ry
Penicillinase Æ β-lactamase

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by e
© lin
APPL. MICROBIOL. 1963; 11: 122-127
n
O
ID
M
C
ES

JAMA. 1964;189:829-834
 ry
TEM-1

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or re
The first plasmid-mediated – β

th tu
lactamase in gram-negatives

au ec
(TEM-1), was described in 1965
from Dr Polixeni Kontomichalou in

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a single strain of E. coli isolated

by e
© lin
during 1964 from a blood culture of
a patient named Temoniera in
n
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Greece, hence the designation
TEM.
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 ry
TEM-2

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Isoelectric

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point

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TEM-1 pI 5.4

au ec
1965

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Gln39→Lys

by e
TEM-2 pI 5.6

© lin
1970
n
O
Identical biochemical properties
ID
M
C
ES

Sutcliffe, J.G. 1978. Nucleotide sequence of

the ampicillin resistance gene of Escherichia
coli plasmid pBR322. Proc. Natl. Acad. Sci.
USA 75:3737-3741.
 MICs (mg/L) for ESBL- producing E. coli

ry
ra
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R- TEM-1+ CTX-1

or re
Amoxicilin 2 1024 >2048

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Piperacillin 1 128 256

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Piperacillin/ Tazobactam 4mg/L 0.5 1 2

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Cefotaxime 0.03 0.03 8

by e
© lin
Ceftazidime 0.25 0.5 16

Aztreonam
n 0.06 0.12 8
O
Cefoxitin 4 4 8
ID

Imipenem 0.12 0.12 0.12

M

Meropenem 0.03 0.03 0.03

C
ES

Sirot D et al. 1987. Transferable resistance to third-

generation cephalosporins in clinical isolates of
Klebsiella pneumoniae: identification of CTX-1, a
novel beta-lactamase. J Antimicrob Chemother;
20:323-34
 ry
ra
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or re
This enzyme may be related
to or derived from the TEM

th tu
enzyme, since an intragenic
probe of the TEM-1 gene

au ec
hybridized with a fragment of
the plasmid carrying CTX-1

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by e
© lin
Fragment patterns of plasmids harbouring CTX-1.
n
Sources of plasmids in E. coli transconjugants were as follows.
O
Lane 1: E. coli
Lane 2: K. pneumoniae
ID

Lane 3: C. freundii
Lane 4: E.aerogenes
M

Lane 5: P. morganii
Lane 6: S. marcescens
C

Lane 7: lambda DNA digested with EcoRI;

ES

Lane 8: pBR322 digested with RsaI.

 ry
TEM-3

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Activity vs

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3rd gen cephs

th tu
TEM-1

au ec
1965

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Gln39→Lys

by e
TEM-2

© lin
1970
n
O
Gln39→Lys Glu104→Lys Gly238→Ser
CTX-1/
ID

TEM-3
M

1987
C
ES
 ry
Extended-spectrum β-lactamases

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ib
Definition:

L
ESBLs are β-lactamases of functional group 2be

or re
or molecular class A that:

th tu
au ec
™Hydrolyze:

L
¾Third generation cephalosporins

by e
¾Monobactams

© lin
•at a rate that is equal to or higher than 10% of
n that for benzylpenicillin
O
™Do not appreciably hydrolyse cephamycins
(cefoxitin or cefotetan) or carbapenems
ID

™Are inhibited by beta-lactamase inhibitors

M

such as clavulanic acid

C
ES
MICs (mg/L) for ESBL- producing E. coli

ry
ra
L ib
R- TEM-1+ TEM-3+ TEM-10+

or re
Amoxicilin 2 1024 >2048 1024

th tu
Piperacillin 1 128 256 >128

au ec
Piperacillin/ 0.5 1 2 2
Tazobactam 4mg/L

L
Cefotaxime 0.03 0.03 8 0.25

by e
© lin
Ceftazidime 0.25 0.5 16 128

Aztreonam
n0.06 0.12 8 64
O
Cefoxitin 4 4 8 4
ID

Imipenem 0.12 0.12 0.12 0.12

M

Meropenem 0.03 0.03 0.03 0.03

C
ES
 ry
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or re
th tu
au ec
L
by e
© lin
n
O
ID
M
C
ES

The Gln39Lys substitution

does not contribute
to the ESBL phenotype
BRADFORD P., CLINICAL MICROBIOLOGY REVIEWS, 2001, 933-951
 ry
Mechanism of action

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or re
β-lactamases

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au ec
L
Serine Enzymes Metallo-Enzymes

by e
Active site

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n
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ID
M
C
ES
 ry
Mechanism of action (Serine based)

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f β-Lactamases catalyse the hydrolytic cleavage of the β-lactam ring of

or re
penicillins, cephalosporins and monobactams

th tu
f The products of fragmentation lack antibiotic activity.

au ec
L
Active

by e
enzyme f The serine residue irreversibly reacts

© lin
with the carbonyl carbon of the
β-lactamase n lactam ring, resulting in an open ring
O
Inactive
penicilloate (inactive lactam) and regenerating
the lactamase.
ID

f Reacts with Penicillins

M

cephalosporins
monobactams
C
ES
 ry
Mechanism of action (Metallo –β- lactamases)

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ib
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or re
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f A divalent transition metal ion,
most often zinc, linked to a

au ec
histidine or cysteine residue or

L
both, reacts with the carbonyl

by e
group of the amide bond.

© lin
f React with penicillins
n
cephalosporins
O
carbapenems
not monobactams
ID
M
C
ES
 ry
Synthesis and mode of transfer

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The synthesis of lactamases is:

or re
™ chromosomal (constitutive)

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™ Pseudomonas aeruginosa

au ec
™ plasmid mediated (inducible)

L
™ Aeromonas hydrophila

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™ Staphylococcus aureus.

© lin
The genetic environment of the β-lactamase
n
(bla) gene dictates whether the β-lactamases
O
are produced in a constitutive or inducible manner.
ID

Plasmids are a major cause of bacterial resistance spreading, as they can be

M

transferred between
C

This transferability is responsible for many

™Gram negative bacteria outbreaks of resistance, especially when
Æ by conjugation
ES

appropriate infection control measures are

™Gram positive bacteria breached in hospital settings.
Æ by bacterial viruses called transducing phages
ES
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M
ID
O
n
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L
Location of β-lactamases

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ry
 ry
β-lactamase classification

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fMolecular classification scheme

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f The simplest classification scheme

th tu
f Based upon amino acid sequence similarity

au ec
f β-lactamases can be divided into four evolutionary distinct

L
molecular classes (A, B, C and D), each with distinct

by e
sequence motifs

© lin
fFunctional classification scheme
n
O
f According to the substrate profile, inhibitor profiles, and
ID

physical characteristics such as molecular weight and

isoelectric point
M

f According to the molecular structure and

C
ES

f According to the nucleotide sequence

 ry
Ambler RP. The structure of beta-lactamases.

ra
Philos Trans R Soc Lond B Biol Sci 1980; 289:321-31

ib
Class A: serine enzymes

L
¾ Staphylococcus aureus PC1 penicillinase

or re
¾ All previously reported serine β-lactamases with extensive sequence homologies with

th tu
each other and with a preference for penicillin substrates are grouped as class A
enzymes.

au ec
Class B: Zinc-requiring enzymes

L
¾ Bacillus cereus β-lactamase II

by e
© lin
Class C: AmpC enzymes from gram-negative bacteria
n
¾ The chromosomally encoded β-lactamases of many species of Enterobacteriaceae have
extensive sequence homologies. They therefore constitute a third group of β-
O
lactamases (class C) that are serine enzymes but probably have an evolutionary origin
ID

different from that of serine penicillinases. (Jaurin & Grundstrom, 1981)

Class D: Oxacillin-hydrolyzing (OXA-type) enzymes
M

¾ Hydrolysis rates for cloxacillin and oxacillin higher than that for benzylpenicillin
C

¾ Were placed in a separate molecular class from the other serine β-lactamases at 1987
ES

from Huovinen et al.

¾ The OXA enzymes of A. baumannii and P. aeruginosa represent the most structurally
diverse and rapidly growing group of beta-lactamases
 ry
Ambler’s Molecular classification system

ra
L ib
PC1 β-lactamase of Staphylococcus aureus

or re
TEM, SHV, CΤΧ-Μ plasmid-β-lactamases

th tu
Inducible cephalosporinases from Klebsiella, P.vulgaris, B.fragilis

au ec
B Metallo-β-lactamases

L
by e
C © lin
AmpC enzymes from gram-negative bacteria; MIR-1
n
O
ID

D OXA-type enzymes
M
C
ES
 ry
Ambler Molecular classification system

ra
L ib
or re
β-lactamases

th tu
au ec
L
Active site Serine Enzymes Metallo-Enzymes

by e
© lin
n
O
ID

Amino acid sequence Α C D B

M
C
ES
 ry
Functional classification of β-lactamases

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Spectrum of antimicrobial substrate profile

or re
Enzyme inhibition profile

th tu
au ec
Enzyme net charge (pI)
Hydrolysis rate (vmax)

L
by e
Binding affinity (km)

© lin
Isoelectric focusingn
Protein molecular weight
O
Amino acid composition
ID
M
C
ES
 BMJ vol. 327, 22 November 2003

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Functional classification of lactamases

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L ib
Year Author Basis of classification of lactamases

or re
1968 Sawai et al Cephalosporins versus penicillins as substrates

th tu
1973 Richmond and Sykes Expanded substrate profile

au ec
Suggested five major groups (Ia-d, II, III, IV, V)
Differentiate plasmid mediated β-lactamases on

L
1976 Sykes and Matthew
the basis of isoelectric focusing

by e
© lin
1981 Mitsuhashi and Inoue Add the category “cefuroxime hydrolysing
nlactamase”
O
1989 Bush Expanded the substrate profile
Add the reaction with EDTA
ID

Correlate functional and molecular classification

M

1995 Bush, Jacoby & Medeiros Expanded the Bush scheme

Used biochemical properties, molecular
C

structure, and nucleotide sequence

ES

Suggested classification into four groups (1-4) on

the basis of the spectrum of activity and other
functional characteristics
 Classification schemes for bacterial

ry
β-lactamases

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or re
th tu
au ec
L
by e
© lin
n
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ID
M
C
ES

Bush K. Characterization of beta-lactamases. Antimicrob Agents Chemother 1989; 33: 259—263.

 Functional classification

ry
«Bush, Jacoby and Medeiros»

ra
L ib
f Four major groups of enzymes are defined by their substrate and

or re
inhibitor profiles:

th tu
f group 1: cephalosporinases that are not well inhibited by clavulanic acid;

au ec
f group 2: β-lactamases that are generally inhibited by active site-directed β-
lactamase inhibitors and that belong to molecular classes A or D;

L
f group 3: metallo β-lactamases that hydrolyze penicillins, cephalosporins, and

by e
carbapenems and are poorly inhibited by classical β-lactamase inhibitors except

© lin
EDTA and p-chloromercuribenzoate (pCMB)
f group 4: penicillinases that are not inhibited by clavulanic acid
n
O
Functional characteristics have been correlated with molecular structure
ID

in a dendrogram for those enzymes with known amino acid sequences.

M
C

(Bush, Jacoby & Medeiros 1995)

ES
 Classification schemes for bacterial

ry
β-lactamases

ra
ib
Ambler Bush Hydrolytic activity Inhibited by Representative enzymes

L
1960 et al
1995

or re
Penicillin Cefotaxime Imipenem Clavulanic acid EDTA

th tu
Α 2a +++ - - ++ - Penicillinases from Gram(+) bacteria

au ec
2b +++ - - ++ - ΤΕΜ-1, ΤΕΜ-2, SHV-1
2be +++ ++ - ++ - TEM- και SHV-type, CTX-M-type,

L
other ESBL
2br +++ - - - - IRT

by e
© lin
2c ++ - - + - PSE-1, PSE-3, PSE-4
2e ++ ++ - ++ - Inducible cephalosporinases from
n Proteus vulgaris
O
2f ++ + ++ + - NMC-1 from E.cloacae, Sme-1 from
S.marcecens
ID

C 1 ++ + - - - AmpC enzymes from Gram (-)

bacteria; MIR-1, LAT-1
M

D 2d ++ - - ± - OXA-1 to OXA-95
B 3 ++ ++ ++ - + L1 from Xanthomonas maltophilia,
C

CcrA from Bacteroides fragilis

ES

Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular
structure. Antimicrob Agents Chemother 1995;39: 1211—1233.
 ry
Total No of β- Lactamases :
>890

ra
KPC

ib
(n=11)
Today: ~500

L
TEM: 188 MβL
SHV: 138 (n=68)

or re
CTX-M: 123

th tu
au ec
Today: 132
CMY-type and TEM-1 ESBL

L
other AmpC-type Today: 227
β-lactamases 18 ESBL activity

by e
CTX-M-1 47 Carbapapene-

© lin
mase activity
n Amp-C
Penicillinases
O
OXA-1
ID

1940s 1950s 1960s 1970s 1980s 1990s 2000s

M
C
ES

Evolution of resistance to β-lactam antibiotics. In parenthesis, the number of the

enzymes reported until November 10, 2011 (http://www.lahey.org/Studies/)
Increase in numbers of group 1, 2, and 3 β-lactamases

ry
from 1970 to 2009

ra
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or re
th tu
au ec
L
by e
© lin
n
O
ID
M
C
ES

Bush K, Jacoby GA. Updated Functional

Classification of β-Lactamases. Antimicrob Agents
Chemother. 2010 March; 54(3): 969–976.
 ry
http://www.lahey.org/Studies

ra
L ib
β-Lactamase Classification and Amino Acid Sequences for TEM,

or re
SHV and OXA Extended-Spectrum and Inhibitor Resistant Enzymes

th tu
George Jacoby Karen Bush

au ec
Lahey Clinic Indiana University Bloomington

L
Burlington, MA 01805 Bloomington, IN 47405

by e
Phone: (781) 744-2928 Phone: (812) 855-1542

© lin
Fax: (781) 744-5486 n Fax:
O
Email: george.a.jacoby@lahey.org Email: karbush@indiana.edu
ID
M
C
ES
ES
C
M
ID
O
n
© lin
by e
L
au ec
th tu
or re
L ib
ra
ry
Proposed β-lactamase classification by Giske et al, 2009

ry
ra
ib
Expands the definition of

L
ESBL to other clinically

or re
important acquired β-
lactamases with activity

th tu
against extended-spectrum

au ec
cephalosporins and/or

L
carbapenems

by e
ESBLA ESBLM ESBLCARBA

© lin
n
O
Class A ESBLs

Μiscellaneous
ID

ESBLs
M
C

ESBLs active against

carbapenems
ES
 ry
ra
L ib
or re
th tu
au ec
L
by e
The term ESBL

© lin
historical definition: differentiate ESBLs from their parental
enzymes that did not hydrolyse extended-spectrum β-lactams.
n
Clinical definition: ESBL-producing organisms are resistant to
O
extended-spectrum cephalosporins and monobactams, but can
be treated successfully with carbapenems.
ID

► Having a category ESBLCARBA, for organisms where

carbapenems should not be used, is inherently unclear and
M

potentially confusing to the practicing clinician.

► Furthermore, the easily understood term carbapenemase
C

defines this functional category so that the designation

ES

ESBLCARBA is unnecessary.
ES
C
M
ID
O
n
© lin
by e
L
au ec
th tu
or re
L ib
ra
ry
Expanded classification schemes for bacterial β-lactamases
Bush & Jacoby 2010

ry
ra
Classification Distinctive Inhibited by Characteristics Representative

ib
substrate(s)
Bush- Bush- Ambler CA EDTA enzyme(s)
Jacoby Jacoby- (1980) or

L
a
(2010) Medeiros TZB

or re
(1995)
1 1 C Cephalosporins - - Greater hydrolysis of E. coli AmpC, P99,

th tu
cephalosporins than ACT-1, CMY-2,
benzylpenicillin; FOX-1, MIR-1

au ec
hydrolyzes cephamycins

L
b
1e NI C Cephalosporins - - Increased hydrolysis of GC1, CMY-37
ceftazidime and often

by e
other oxyimino-β-lactams

3a 3 B (B1)
© lin
Carbapenems
n - + Broad-spectrum
hydrolysis including
IMP-1, VIM-1,
CcrA, IND-1
O
carbapenems but not
monobactams
ID

B (B3) L1, CAU-1, GOB-1,

M

FEZ-1
C

Preferential hydrolysis
3b 3 B(B2) Carbapenems - + of carbapenems CphA, Sfh-1
ES

NI 4 Unknown
a CA, clavulanic acid; TZB, tazobactam.
b NI, not included
 Classification Distinctive Inhibited by Characteristics Representative

ry
Bush- Bush- Ambler substrate(s) CA or EDTA enzyme(s)
a
Jacoby Jacoby- (1980) TZB

ra
(2010) Medeiros
(1995)

ib
2a 2a A Penicillins + - Greater hydrolysis of benzylpenicillin PC1

L
than cephalosporins
2b 2b A Penicillins, + - Similar hydrolysis of benzylpenicillin TEM-1, TEM-2,

or re
Cephalosporins and cephalosporins SHV-1
2be 2be A Extended-spectrum + - Increased hydrolysis of oxyimino-- TEM-3, SHV-2,

th tu
cephalosporins, lactams (cefotaxime, ceftazidime, CTX-M-15, PER-1,
monobactams ceftriaxone, cefepime, aztreonam) VEB-1

au ec
2br 2br A Penicillins - - Resistance to clavulanic acid, TEM-30, SHV-10
sulbactam, and tazobactam

L
2ber NI A Extended-spectrum - - Increased hydrolysis of oxyimino— TEM-50
cephalosporins, lactams combined with resistance to

by e
monobactams clavulanic acid, sulbactam, and

© lin
tazobactam
2c 2c A Carbenicillin + - Increased hydrolysis of carbenicillin PSE-1, CARB-3
2ce NI A Carbenicillin,
cefepime
n + - Increased hydrolysis of carbenicillin,
cefepime, and cefpirome
RTG-4 (CARB-10)
O
2d 2d D Cloxacillin ± - Increased hydrolysis of cloxacillin or OXA-1, OXA-10
oxacillin
ID

2de NI D Extended-spectrum ± - Hydrolyzes cloxacillin or oxacillin and OXA-11, OXA-15

cephalosporins oxyimino--lactams
M

2df NI D Carbapenems ± - Hydrolyzes cloxacillin or oxacillin and OXA-23, OXA-48

carbapenems
C

2e 2e A Extended-spectrum + - Hydrolyzes cephalosporins. Inhibited CepA

ES

cephalosporins by clavulanic acid but not aztreonam

2f 2f A Carbapenems ± - Increased hydrolysis of KPC-2, IMI-1,
carbapenems, SME-1
oxyimino--lactams, cephamycins
a CA, clavulanic acid; TZB, tazobactam. b NI, not included
Distribution of β-lactamases according to functionality

ry
ra
ib
2, 2a, 2b, 2c, and 2d 2df, 2f, and group

L
β-lactamases 3 β-lactamases

or re
th tu
au ec
L
by e
1, 1e, and 2e

© lin
enzymes
n
O
ID
M

2be, 2ber, and 2de

β-lactamases
C
ES

Karen Bush. Current Opinion in Microbiology 2010; 13: 558-564

Major families of β-lactamases of clinical importance

ry
ra
ib
Extended-spectrum β-lactamases

L
f TEM, SHV & CTX-M type

or re
fAmbler’s class Α
fBush et al group 2be

th tu
AmpC

au ec
f Species-specific AmpC chromosomal enzymes

L
f Plasmid-encoded AmpC cephalosporinases

by e
fAmbler’s class C

© lin
fBush et al group 1

Carbapenemases
n
O
f Serine carbapenemases
f
ID

Metallo-β-lactamases
fAmbler’s class Α, Β και D
M

fBush et al group 2f, 2df και 3a

C
ES
 Major families of β-lactamases of clinical importance

ry
ra
Enzyme Molecular Functional group or No of enzymes Representative enzymes
Family class subgroup

ib
TEM A 2b, 2be, 2br, 2ber 183

L
A 2b 14 TEM-1, TEM-2, TEM-13
A 2be 82 TEM-3, TEM-10, TEM-26

or re
A 2br 36 TEM-30 (IRT-2), TEM-31 (IRT-1), TEM-163

th tu
A 2ber 11 TEM-50 (CMT-1), TEM-158 (CMT-9)
SHV A 2b, 2be, 2br 137

au ec
A 2b 31 SHV-1, SHV-11, SHV-89
A 2be 45 SHV-2, SHV-3, SHV-115

L
A 2br 6 SHV-10, SHV-72

by e
CTX-M A 2be 121 CTX-M-1 to CTX-M-123

© lin
PER A 2be 7 PER-1 to PER-7
VEB A 2be n 7 VEB-1 to VEB-7
GES A 2f 16 GES-2 to GES-17
O
KPC A 2f 10 KPC-2 to KPC-11
SME A 2f 3 SME-1, SME-2, SME-3
ID

IMP B 3a 29 IMP-1 to IMP-29

VIM B 3a 27 VIM-1 to VIM-27
M

NDM B 3a 2 NDM-1, NDM-2

IND B 3a 8 IND-1, IND-2, IND-2a, IND-3 to IND-7
C

CMY C 1, 1e 86 CMY-1 to CMY-86

ES

OXA D 2d, 2de, 2df 203

D 2d 5 OXA-1, OXA-2, OXA-10
D 2de 16 OXA-11, OXA-14, OXA-15
D 2df 48 OXA-23 (ARI-1), OXA-51, OXA-58
Extended-spectrum β-lactamases (ESBL)

ry
– Bush group 2be

ra
L ib
TEM-, SHV- family

or re
Arose by amino acid substitutions in TEM-1, -2 (82 enzymes) or SHV-1 (45

th tu
enzymes).

au ec
First recognized in 1983. Their emergence and rapid dissemination have
been responsible for numerous outbreaks of infection throughout the world.

L
Mostly found in K. pneumoniae and E. coli (also in C. freundii, Enterobacter

by e
aerogenes, and Serratia marcescens).

© lin
Mostly encoded by large plasmids (up to 100 kb and even more) that are
n
transferable from strain to strain and between bacterial species.
O
Hydrolyse penicillins, cephalosporins (except cefoxitin), monobactams.
ID

Do not hydrolyse carbapenems (IMP, MEM, ERT)

M

Inhibited by clavulanic acid

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ES
Extended-spectrum β-lactamases (ESBL)

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– Bush group 2be

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CTX-M-1 έως CTX-M-123

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Derivatives of chromosomal genes resident in members of the genus
Kluyvera.

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Clustered in six groups (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, CTX-M-25,

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CTX-M-45) with similarity at the amino-acid sequence level.

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The first CTX-M-type enzyme of clinical origin, CTX-M-1, was described in

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enterobacterial strains isolated in Europe in the late 1980s

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Within a decade, the CTX-M β-lactamases became the predominant ESBL
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family in many medical centres such that they have largely replaced most of
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the TEM- and SHV-derived ESBLs throughout the world.
Most (but not all) CTX-M enzymes hydrolyze cefotaxime more readily than ceftazidime. Many
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hydrolyze cefepime as well.

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Inhibited by tazobactam at least an order of magnitude better than by clavulanic acid

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AmpC enzymes

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f Class C cephalosporinases, Group 1 β-lactamases

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f Are encoded primarily in the chromosome of many species of the

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Enterobacteriaceae

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f Hydrolytic activity
f 1st gen cephalosporins –rapid

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f 2nd and 3rd gen cephalosporins –slow but kinetically efficient

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f 4th gen cephalosporins –slow, kinetically inefficient

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f Carbapenems –nearly stable

Basal in:
n Inducible in:
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E. coli & shigellae Enterobacter spp.
C. freundii
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M. morganii
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Serratia spp.
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P. aeruginosa
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plasmid-mediated AmpC β-lactamases

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f Increasingly reported worldwide (since 1989)

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f Escaped from the chromosome of natural AmpC-producing

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species of the Enterobacteriaceae

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f Are categorized into six groups

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Group Origin Homology Members

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CMY-2 Citrobacter freundii 96% 85

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MIR-1 & ACT-1 n Enterobacter spp 98-99% 15
DHA Morganella morganii 99% 8
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ACC-1 Hafnia alvei 99% 4
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FOX-1 Aeromonas caviae 99% 10

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CMY-1 (also called MOX-1) A.hydrophila 82% 8

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EUCAST breakpoints for Enterobacteriaceae

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Cephalosporins MIC (mg/L) Disk (µg) Diameter (mm)

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S≤ R> S≥ R<
Cefadroxil 16 16 30 12 12

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Cefuroxime 8 8 30 18 18

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Cefotaxime 1 2 5 21 18

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Ceftriaxone 1 2 30 23 20

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Ceftazidime 1 4 10 22 19

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Aztreonam 1 4 30 27 24
Cefepime 1
n 4 30 24 21
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Cefoxitin NA NA 19 19
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Detect all clinically important resistance mechanisms (ESBL and plasmid

mediated AmpC).
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Strains susceptible or intermediate to 3rd or 4th generation

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cephalosporins should be reported as found.

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ESBL detection and characterization is recommended or mandatory for

infection control purposes.
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CLSI breakpoints for Enterobacteriaceae

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Cephalosporins MIC (mg/L) Disk (µg) Diameter (mm)

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S≤ R> S≥ R<

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Cefazolin 1 2 NA NA

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Cefuroxime 8 16 30 18 15

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Cefotaxime 1 2 30 26 23
Ceftriaxone 1 2 30 23 20

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Ceftazidime 4 8 30 21 18

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Aztreonam 4 8 30 21 18

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Cefepime 8
n 16 30 18 15
Cefoxitin 8 16 30 18 15
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Eliminate the need to perform ESBL screen and confirmatory tests for
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making treatment decisions.

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Detection and confirmation are less accurate when multiple enzymes are
present.
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MIC correlates better with clinical outcome than knowledge of resistance

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mechanisms.
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CONFIRMATORY TESTS FOR ESBLS

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Double disc tests.

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™ Cefotaxime, ceftazidime, cefepime and aztreonam disks 30 μg are
applied 25-30 mm away from a co-amoxiclav 20+10 μg disk.

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When the zone of either cephalosporin is expanded by the clavulanate ÆESBL
production

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ƒ cheap
ƒ critical disc spacing Ævaries with the strain, some producers may

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be missed.
Combination disc methods.

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™ Cefotaxime or Ceftazidime 30μg disks

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™ Cefotaxime or Ceftazidime plus clavulanate 30+10μg disks

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Zone diameters of ≥5 mm in the presence of the clavulanate Æ ESBL
production
ƒ cheap
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ƒ do not require critical disc spacing
Etest ESBL strips.
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™ Ceftazidime or Cefotaxime 0.25-128 μg/ml

™ Ceftazidime or Cefotaxime 0.25-128 μg/ml + clavulanate 0.25/4-128/4
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μg/ml
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MIC decline of ≤ 3 in the presence of the clavulanate Æ ESBL production

ƒ Follow the manufacturer’s instructions
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ƒ Accurate and precise

ƒ More expensive than combination discs.
AmpC DETECTION

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f Induction by FOX f Inhibition by 3-aminophenylboronic
(Reduction in CAZ zone diameter) acid or cloxacillin

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FOX

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f Cover leaf test
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n f Εtest Cefotetan /
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Cefotetan+Cloxacillin
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ID
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n
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And now its time for Carbapenemases
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ID

Karyatides-New Acropolis Museum

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Thank you for your attention

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