1. IN THE NAME OF GOD
IN THE NAME OF GOD
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2. january 2013
Hamadan University of Medical Sciences
Extended Spectrum Beta-Lactamases (ESBLs)
Supervisor: Dr. Yousefi
By: Seyed Mostafa Hosseini
e.mail: smhoseiny88@yahoo.com
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Betalactam Antibiotics
Penicillins Cephalosporins Monobactams Carbapenems
Advantages
High effectiveness
Low cost
Minimal side effects

4. MECHANISMS OF RESISTANCE TO Β-LACTAM
ANTIBIOTICS
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1. Inactivation of the β–lactam antibiotic by an enzyme (β–lactamase)
2. Production of newer PBP with decreased affinity to the antibiotic
3. Decreased permeability of the antibiotic through the cell envelope due to
altered porin channels
4. Active efflux pumpes that throw the antibiotic out of the cell

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One of the most important resistance
mechanism is producing
Betalactamase
enzymes
Extended spectrum beta-lactamases
(ESBLs )
This enzymes belong to group 2be, 2d of
functional classification and class A , D of
molecular classification

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Classification of ESBLs:
Main Groups:
Other ESBLS:
 OXA
 PER
 VEB
 TLA
 BES
 BEL
 GES
 SFO

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TEM was first discovered in E coli in a patient named
Temoniera in Greece
TEM-1 is the most prevalence in this group
TEM-2 is a derivative of TEM-1, which possesses an amino acid
substitution
Phenotypic variation of ESBLs is caused by alteration of amino
acid sequence at the active site
TEM-type β-lactamases are most often found in E. coli and K.
pneumoniae
Until May 2011, 190 types of TEM group have been reported

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SHV-1 was described in 1972 by Pitton in a Klebsiella
pneumoniae isolate pit2
SHV refers to “sulfhydryl variable” of active site
All SHV variants described to date are derived from
blaSHV-1 with one to seven amino acid substitutions
There have been reported for 141 types of SHV at the
end of May 2011

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Plasmid-mediated ESBLs CTX-M,
hydrolyze cefotaxime over than ceftazidime
(cefotaximase, Munich)
The CTX-M enzymes originated from
Kluyvera spp Of environmental, for example
K.ascorbata and K. georgina are the
progenitors of the CTX-M2 and CTX-M8.
Five group of CTX-M are: CTX-M1, -2, -8, -9
and -25.
 CTX-M are associated with mobile elements
such as ISEcp-1 CTX-M 1, 2, 3, 9, 13, 14,
15, 17, 19, 20, 21
At the end of May 2011, 120 types of
enzymes have been reported

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They have high hydrolytic activity against
oxacillin and cloxacillin and the poorly inhibited
by clavulanic acid
Not all OXA enzymes are ESBLs; ESBLs are
OXA-11, -14, -16, -17, -18, -19, -28, -31, -32, 35
and -45
Several of the OXA-type ESBLs have been
derived from OXA-10 (OXA-11, -14, -16, and
17)

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 Inhibitor-resistant β-lactamases are not ESBLs, but they are
often discussed with ESBLs because they are also derivatives
of the classical TEM- or SHV-type enzymes
The amino acid substitutions in IRTs are different from
those that are in ESBL phenotype. This variants are called
Complex Mutant of TEM (CMT) that are placed in Bush
subgroup 2ber
IRTs variants are resistant to inhibition by clavulanic acid
and sulbactam but They remain susceptible to inhibition by
tazobactam
IRT enzymes have also been found among SHV mutant and
K.pneumoniae isolates. Seven SHV enzymes (-10, -26, -49,
-56, -72, -84, -107) with IRT phenotype are known

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PER-1 type β-lactamase was reported in 1991 from
P. aeruginosa isolate in Turkey
 PER-type enzymes give high level resistance to
ceftazidime, cefotexim and aztreonam but low level
resistance to other cephalosporins and inhibited by
clavulanic acid but not by tazobactam
 PER-type enzymes have25% -27 % homology to
TEM and SHV

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VEB-1 was first described in an E coli strain
isolated in a French hospital from the pus of a 4-month
old Vietnamese boy in 1996
It confers high level resistance to ceftazidim,
cefotaxim and aztreonam, and inhibited by clavulanic
acid
The gene encoding VEB-1 was found to be plasmid
mediated; such plasmids also confer resistance to non β-
lactam antibiotics

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Which was isolated from Chryseobacterium meningosepticum
Which was identified in an E. coli isolate from a patient in
Mexico in 1993
Was isolated from a S. marcescens strain from a hospital in
Brazil in 1996. It confers a high level of resistance to
aztreonam and a higher level of resistance to cefotaxime than
to ceftazidime and well-inhibited by clavulanate than by
tazobactam

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Was identified in a P. aeruginosa strain isolated in Belgium in
2004. It significantly hydrolyses most expanded-spectrum
cephalosporins and aztreonam, but not cephamycin or
carbapenem. Its activity is well-inhibited by clavulanate but
poorly so by tazobactam
It is another uncommon ESBL enzyme that is not closely
related to any other plasmid-mediated β-lactamase but does
show 36% homology to a carbenicillinase from Proteus
mirabilis
which is highly related to a class A β-lactamase from Serratia
fonticola, high level of resistance to cefotaxime than to
ceftazidime and well-inhibited by clavulanate

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First reports of ESBLs in the USA in the late 1980s were
reported with TEM-type major enzymes appear to be the TEM
and SHV types, with a minimal appearance of CTX-M types
During the 1980s and 1990s, TEM and SHV were the predominant
ESBL type. Currently, CTX-M and TEM are the main types
Rates of ESBLs in South America rank amongst the highest in
the world, with CTX-M dominant

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In Asia, high rates of ESBL producing Enterobacteriaceae are seen
The first reports from Japan and Taiwan suggest the SHV type
played a key role early on but, just like in mainland Europe, the
rise of the CTX-M genotype has made it the preeminent enzyme,
with national and regional variations
CTX-M genotype appears to be the most common type in North
Africa. There have also been reports of CTX-M K. pneumoniae in
Kenya and SHV and TEM-types in South Africa

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1. Hospital stay of more than seven days
2. Exposure to nosocomial isolates
3. Transfer from another health care facility
4. Central venous catheterization
5. Female sex
6. Hemodialysis
7. Diabetes mellitus
8. Antibiotic use (third and fourth generation cephalosporins)
for more than a week
9. Travel to endemic countries

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Laboratory
detection of eSbLS

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PhenotyPic methodS
Screening
teStS:
A. Disk Diffusion method:
The screening test must be performed on Mueller Hinton
agar on standardized inoculum (0.5 McFarland turbidity)
and diameter of the zone of inhibition must be carefully
measured after 16-18 hours of incubation at 35-37ºC.

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For E. coli, K. pneumoniae, and K. oxytoca, MIC > 8
µg/ml for cefpodoxime or MIC > 2 µg/ml for ceftazidime,
aztreonam, cefotaxime, or ceftriaxone is suggestive of
ESBL production. For P. mirabilis, MIC > 2 µg/ml for
cefpodoxime, ceftazidime, or cefotaxime is suggestive of
ESBL production.
B. Microbroth dilution
method:
C. Chromogenic
selective medium:
Brilliance ESBL agar (Oxoid, UK)
ChromID ESBL agar (bioMe´rieux, France)

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eSbL confirmatory methodS:
1. Combined disk:
CAZ/CLA – 22 mmCAZ/CLA – 22 mm
CAZ – 11 mmCAZ – 11 mm
22 –11 = > 5mm ESBL

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2. Double Disk Synergy
Test (DDST):
Double Disk Diffusion Test (DDDT)
cefotaximecefotaxime Amoxyclav
(amoxicillin/clavula
nic acid 30/10 µg)
Amoxyclav
(amoxicillin/clavula
nic acid 30/10 µg)

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3. Three dimensional (3D)
tests:

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Commericailly available Method for ESBL Detection:
1. E test1. E test

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positive ESBL E test
Unreadable Etest ESBL screen test. The MIC of ceftazidime, which should
be read on the end of the strip marked “TZ,” cannot be determined because
the inhibition ellipse is distorted by the action of clavulanic acid diffusing
from the opposite end of the strip.

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Other Commericailly Method for ESBL Detection:
1. Sensititre (TREK Diagnostic system)
2. MicroSan (Siemens Medical Solution
Diagnostics)
3. Phoenix (BD Diagnostic system)
4. Vitek (BioMerieux)

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Genotypic methods:
1. Conventional PCR: Materials:
DNA template Forward Primer buffer10x
Taq Polymerase Reverse Primer dNTP 10mM
Primery Denaturation 94ºC, 5min
Final Denaturation 94ºC, 1min
30-35 Annealing 50ºC, 1min
Primery Extension 72ºC, 1 min
Final Extension 72ºC, 8 min

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2. Mutiplex PCR
3. PCR-Restriction Fragment Length Polymorphism (PCR-RFLP):
The amplicons generated following PCR are digested by specific
restriction endonucleasesand the fragments that are generated are
seprated by gel electrophoresis. The size of fragment indicate point
mutations whithin the structural gene.
4. Ligase chain reaction (LCR):
Using four different sets of primers. The target DNA sequence
after denaturation, the primers hibridize to their complementary
strands. The thermostable ligase enzym ligates only the primers
that are perfectly complementary to their target sequence. The
LCR product is detected by an enzymatic reaction using
NADPH- alkalin phosphatase.

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treatment options aGainst esBL producers
Carbapenems
Piperacillin-tazobactam
Fluoroquinolones
Cefepime

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infection control
Hand washing by hospital personnel
 Isolation of patients colonised or infected with ESBL
producers
Clinical and bacteriological surveillance of patients
admitted to intensive care units and antibiotic cycling,
as well as policies of restriction, especially on the
empirical use of broad spectrum antimicrobial agents
such as the third and fourth generation cephalosporins
and imipenem.

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thank you