LECTURES SERIES ESBLs

OVERVIEW OF EXTENDED
SPECTRUM BETA-LACTAMASES
(ESBLs)
DEPARTMENT OF PHARMACEUTICS AND
PHARMACEUTICAL MICROBIOLOGY
USMANU DANFODIYO UNIVERSITY, SOKOTO
NUHU, Tanko
30th June, 2016

Presentation Outline
Introduction
Beta-lactam antibiotics
Antimicrobial resistance
Beta-lactamases
Other definition of ESBLs
Methods of detection of ESBL production
July 16 2Overview of ESBLs Nuhu Tanko

Introduction
• The bactericidal effect of beta-lactam antibiotics
involves inhibition of cell wall synthesis.
• This effect occurs through covalent attachment to
PBP, which is a peptidoglycan transpeptidase
enzyme that catalyzes the final steps in cell wall
formation.
• The spectrum and effects of the different beta-
lactams are determined by the PBPs to which
these antibiotics bind.

Brief background of Beta-lactam (BL)
antibiotics
• The 1st semi-synthetic cephalosporins were
introduced in the mid 1960s, and they showed a
somewhat limited Gram-ve effect.
• In 2GC, coverage was expanded to include Gram-ve
bacteria in addition to the Gram+ve effect.
• The 3-GC (oxyimino-beta-lactams) offered extended
coverage of Gram-ve bacteria and even better BL
stability.
• In part, these cephalosporins were developed
because of the discovery of narrow-spectrum beta-
lactamases (e.g., TEM-1), and some of them also
had good oral bioavailability.

• The Gram-ve effect was extended even further in
the 4-GC.
• A better Gram+ve effect was gained in the 5GC,
and this even applied to MRSA, and hence the
5GC are also called MRSA-active
cephalosporins.
• The cephamycins were developed in the 1970s.
• They proved to have the same antimicrobial
effect as the 2GC cephalosporins but were stable
against class A ESBLs.

• The carbapenems, were found to be highly
resistant to enzymatic hydrolysis.
• The 1st carbapenem (imipenem), which had to be
combined with cilastatin to protect it against renal
dehydropeptidase.
• The 2G of carbapenem, are resistant to renal
dehydropeptidase.
• The carbapenems are known to be the ONLY
antibiotics that have some degree of post-
antibiotic effect on infections with Gram-ve.

• The first monocyclic bacterially produced beta-
lactam antibiotics were named monobactams
(e.g., aztreonam).
• Monobactams have a good Gram-ve effect but
no useful Gram+ve effect, and they are stable
towards several beta-lactamases.

The menace called Antimicrobial
Resistance (AMR)
• Bacteria/microbes are remarkably resilient and
have developed ways to resist antibiotics/other
AM drugs.
• AMR is a resistance of microorganism to an AM
that was originally effective for treatment of
infections caused by it.

What are the Mechanism of
Resistance?
 Destruction or inactivation of the antibiotic
 Alteration or protection of the target site.
 Reduction in cell surface permeability.
 Metabolic by-pass.

• AMR can be:
Intrinsic resistance, or
Acquired resistance
• Factors promoting AMR:
Exposure to sub-optimal level of AM.
Exposure to broad spectrum AM.
Exposure to microbes carrying resistant genes.
Lack of hygiene in clinical environment.
Use of antibiotics in food and agriculture.

The Enzymes called Beta-lactamases (BLs)
• The BLs are the collective name of enzymes that
open the BL ring.
• Identified as a penicillinase or cephalosporinase on
the basis of the substrate hydrolysed.
• A water molecule is added to the common BL bond,
and this inactivates the BL antibiotic from penicillin
to carbapenems.
• Clinical effect of such hydrolyzation was not noted
until the beginning of the 1950s, when the first BL-
resistant S. aureus isolates appeared in hospitals.

What are these ESBLs?
• In 1983, Knothe found a single nucleotide mutation
in an SHV that represented the first plasmid-encoded
BL that could hydrolyze the extended-spectrum
cephalosporins in an isolate of K. ozaenae, and this
type was named SHV-2.

• ESBLs are defined as BLs that have the following
characteristics: they are transferable; they can
hydrolyze penicillins, 1st, 2nd, and 3rd GC, and
aztreonam (but not the cephamycins and
carbapenems); they can be blocked in vitro by
BLs inhibitors such as clavulanic acid.
• ESBL production is originally observed in E. coli and
Klebsiella spp., and has now been documented in
other Gram-ve bacilli, including Enterobacter spp.,
Proteus mirabilis and Providencia stuarti.

• Most ESBLs can be divided into three groups, which
are designated the TEM (approx. 200 variants), SHV
(over 140 variants), and CTX-M (approx. 130).
• In the beginning of the ESBL era, the clinical isolates
consisted of the TEMs and SHVs (mainly SHV-2 and
SHV-5).

• Members of the CTX-M group are now the most
common ESBLs worldwide.
• In Germany in 1989, an E. coli isolate that was
resistant to cefotaxime and produced a non-
TEM–non-SHV enzyme, was named CTX-M-1
due to its elevated activity against cefotaxime.

• The CTX-M enzymes can be classified into five
major groups, which are designated CTX-M-1, CTX-
M-2, CTX-M-8, CTX-M-9, and CTX-M-25.
• Each of these includes several plasmid-mediated
enzymes.
• For example, the CTX-M-1 group comprises CTX-
M-15 and several other types.

How can we detect ESBL production
in the laboratory?
• Can be Phenotypic or Genotypic.
• Increasing resistance to 3GC is predominantly
due to the production of ESBLs.
• Accurate laboratory detection is important to
avoid clinical failure due to inappropriate
antimicrobial therapy.

• As a general rule, laboratories should test all isolates
using both ceftazidime (the best indicator for
TEM and SHV-derived ESBLs) and cefotaxime
(the best indicator for CTX-M types).
• Alternative, they can test with cefpodoxime, as a
good indicator for all ESBL types.
• Earlier advice to screen only with ceftazidime is no
longer adequate in view of the emergence of CTX-M
types.

• Any organism showing reduced susceptibility to
cefotaxime, ceftazidime or cefpodoxime should
be investigated for ESBL production.
• Many different techniques exist for confirming
ESBL production but those utilising similar
methodology to standard susceptibility tests are
the most convenient for the routine diagnostic
laboratory.
• These all depend on detecting synergy between
clavulanic acid and the indicator
cephalosporin(s) used in the primary screening.

Each isolate should be considered a potential
ESBL-producer if the test results are as follows:
• Disk diffusion
 Cefpodoxime < 22 mm
 Ceftazidime < 22 mm
 Cefotaxime < 27 mm
 Ceftriaxone < 25 mm
 Aztreonam < 27 mm
• MICs
 Cefpodoxime > 2 µg/ml
 Ceftazidime > 2 µg/ml
 Cefotaxime > 2 µg/ml
 Ceftriaxone > 2 µg/ml
 Aztreonam > 2 µg/ml
July 16 Overview of ESBLs Nuhu Tanko 20

Double disc method
• Here it examines for the expansion of the
cephalosporin’s inhibition zone adjacent to a disc
containing Co-amoxiclav 20 + 10 mg.
• The agar is inoculated with the test organism to
give a semi-confluent growth.
• A ceftazidime 30 mg disc and an Co-amoxiclav
20+10 mg disc are then placed 25 - 30 mm apart,
centre-to-centre.
• This follow an overnight incubation in air at
37°C.

ESBL production is inferred when the zone of inhibition around the ceftazidime disc
is expanded by the clavulanate.
Additional extended-spectrum cephalosporins may be tested concurrently eg
cefotaxime (30 mg), aztreonam (30 mg) and ceftriaxone (30 mg), providing the Co-
amoxiclav disc is placed in the centre of the plate and the distance of 25 - 30 mm
between the cephalosporin and clavulanate-containing discs is observed.
The use of multiple cephalosporins may be helpful as ESBLs other than common
TEM and SHV mutants begin to spread.

Combination disc methods
• The zone of inhibition around a ‘combination’
disc containing the cephalosporin combined with
clavulanic acid is compared to the zone around a
disc containing the cephalosporin alone.
• An expansion of >5 mm or 50% (according to the
particular product and manufacturer’s guideline)
indicate ESBL production.
• Plates of test organisms are inoculated.

• Pairs of discs containing an extended-spectrum
cephalosporin (cefotaxime, ceftazidime or
cefpodoxime) with and without clavulanic acid
are placed on opposite sides of the same
inoculated plate.
• Zones of inhibition are measured following
overnight incubation in air at 37°C.
• The test organism is regarded as an ESBL
producer if the zone of inhibition around the
combination disc is at least 5 mm larger than that
of the cephalosporin alone, or if the zone
diameter is expanded by 50% in the presence of
the clavulanic acid.

CZC
CZ
CTC
CT
Based on CLSI recommendation, an increase in zone size > 5 mm than the zone when
tested alone will be accepted as confirmation of ESBL production.

ESBL Etest
• The detection of ESBLs by Etest is based on a
similar principle to that of the combination disc
method.
• Double-ended strips containing gradients of
cefotaxime or ceftazidime at one end and
cefotaxime or ceftazidime plus clavulanic acid at
the other end are tested in parallel.
• A decrease in MIC of 3 doubling dilutions in the
presence of clavulanate indicates ESBL
production.

Note
• Clinical isolates found to produce ESBLs should
be assumed to be resistant to all extended
spectrum cephalosporins irrespective of the
results of susceptibility testing.
• Not all resistance to 3GC due to ESBL
production - other potent BLs such as AmpC and
K1 enzymes may be responsible.

In Conclusion
• 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.

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LECTURES SERIES ESBLs

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