Extended-Spectrum β-

Lactamases
(ESBLS)
Laboratory Guideline For The
Detection Of This Important Resistance
Treat
Prof. Ellabib MS
Dept. of Medical Microbiology
Medical College
ellabib@dmi.ly
 Introduction to β-Lactamases
What is β-Lactamases ?
Enzymes produced by Bacteria
Inactivate β-Lactam antibiotics
Chromosomal or plasmid-mediated
Rare among gram positive pathogens
Exceptions being Staph. aureus and Enteroccocus
faecalis
Common among gram negative bacteria
Enterobacteriaceae
Inducible, constitutive and /or hyper productive strain
Such as TEM-1 and SHV-1
Introduction to β- Lactamases cont.
 What is Extended-Spectrum β- Lactamases?
 Mutation of TEM-1 AND SHV-1 leading to
 Enzymes hydrolyze β- Lactam antibiotics
 Cause resistant to Oxyimino - cephalosporins
Ceftazidime
 Aztreonam
 Produced by E. coli, Klebsiella pneumoniae
 Other Enterobacteriaceae
 P. aeruoginosa
 Chromosomally mediated or plasmid- mediated
 History to β-Lactamases
 β-Lactamases in gram positive bacteria
 Represented by Penicillinase enzyme
 S. aureus, coagulase negative staphylococci
and Enteroccocus faecalis
 80 to 90% resistant to penicillin's
 Benzylpenicillins
 Aminopenicillins (ampicillin, amoxicillin)
 carboxypenicillins (ticarcillin, carbenicillin)
 Remain sensitive to stable penicillin
 Methicillin, oxacillin
β-Lactamases in gram positive bacteria cont.

Class A enzyme (plasmid mediated)

 Borderline oxacillin resistant
Hyper producer strains of S. aureus
Show resistant to oxacillin
Retain sensitivity with clavulanic acid
Not major problem in other G+ bacteria
Major important resistant in S. aureus is
Methicillin resistant
β-Lactamases in gram negative
Leading cause of resistant
Chromosomal or plasmid
Constitutive or inducible
TEM-1 (E. coli, 1960) Temoniera
Plasmid
SHV-1
Plasmid mediated in E. coli
Chromosomally in most K. pneumoniae
β-Lactamases in gram negative
New β-Lactamases for every new β-Lactam
 Selective pressure
 Over use of new antibiotics
 Select new variant of β-Lactamases resistant
 SHV-2
 First enzyme hydrolysis newer β-Lactam
 Found in K. ozraenae in Germany
 Called ESBLS
 OVER 150 ESBLS were discovered
 Enterobacteriaceae and Ps. aeruginosa
Characterization and Classification of ESBLS

 Most of them has serine at active site

 Class A enzyme
 Hydrolysis penicillin's
 Such as TEM-1, SHV-1 and penicillinase

 Bush, Jacoby and Medeiros

 Classification scheme
 Use biochemical enzyme properties
 Molecular structure
 Nucleotide sequence genes
 Place β-Lactamases into functional groups
Types of ESBLS
 Most of them derived from TEM OR SHV
 Mostly found in E. coli and K. pneumoniae
 Also found in other Enterobacteriaceae

TEM types
 TEM-1
 E. coli
 90% amp Resistant
 H. influenzae and N. gonorrhoeae
 Amp and p resistant
 Hydrolyze penicillins and early cephalosporin's
TEM-2
 Derived from TEM-1
 NO CHANGE IN SUBSTRATE PROFILE
 Tem-3
 First TEM type displayed ESBLS
 Over 90 TEM derivatives described latter
 ESBLS and inhibitory resistant (IRT)
 Derivatives of TEM and SHV types
 Resistant to inhibition by clavulanic acid
 TEM-1 and TEM-2
 Mainly found in E. coli
 Some strain of K. pneumoniae and K. oxytoca
ESBLS and inhibitory resistant (IRT) cont.
 Resistant to sulbactam
 Remain susceptible to tazobactam
 Sulphydryl variable (SHV)
 SHV-1
 K. pneumoniae
 Plasmid mediated
 Amp resistant
 In many strains genes integrated in chromosomes
 Few derivatives of SHV-1
 Serine residue critical for hydrolysis of ceftazidime
 Lysine for cefotaxime
Sulphydryl variable (SHV) cont.
 SHV-10
 Have an IRT
 Majority found in K. pneumoniae
 Citrobacter diversus and E. coli
 P. aeruginosa
 CTX-M
 Plasmid mediated ESBLS
 Hydrolyze cefotaxime over ceftazidime
 Strains of Salmonella enteric serovar Typhimurium
 E. coli and other some species of enterobacter
 Better inhibited by Tazobactam
OXA
 Class D group 2d
 Confer resistant to Amp and cephalothin
 High hydrolytic activity against OX and CLOX
 Poorly inhibited by clavulanic acid
 Found mainly in P. aeruginosa
 PER-1
 Plasmid encoding
 P. aeruginosa and Salmonella enteric serovar Typhimurium
 Acinetobacter baumanii
 Ceftazidime resistant strains in A. baumanii
Laboratory detection of ESBLS
Why ?
 Major leading cause of resistance to β - Lactam
antibiotics
 Increasing prevalence among
Enterobacreriaceae
 Due to its fast Spread amongst various bacteria
 Very Important by infection control committee
 Clinical and therapeutic implication
Methods
1. Double disk diffusion Synergy test (DDST)
2. Broth dilution
3. Etest ESBL strip

 Double disk diffusion (DDST)

 Using Kirby-Bauer disc diffusion (NCCLS)
 Synergy between augmentin+3GC
 Ceftazidime, Cefpodoxime,
 Cefepime, cefotaxime, cefoxitin
 Increased zone with the addition of inhibitor
DOUBLE DISK TEST FOR ESBL CONFIRMATION
l. Introduction
 Cefpodoxime* (can be used alone)
 third generation cephalosporins
 Aztreonam
 are all extremely susceptible to ESBLs
 used as screening agents
 MIC
 disk diffusion
 E. coli, Klebsiella species or Proteus species
 confirmation of ESBL be determined by the
double disk test.
II. Materials
Mueller‑Hinton (MH) agar (90 0r 150) mm
20/10 µg amoxicillin‑ clavulanate disc
30 µ g ceftazidime disc
30 µ g ceftriaxone or cefotaxime disc
30 µ g aztreonam disc
10 µ g cefpodoxime disc (optional)
30 µ g cefoxitin disc
30 µ g cefepime disc
Quality control strain: E. coli ATCC 51446
lll. Procedure
1. Prepare a bacterial suspension of the organism to be
tested that has a turbidity equivalent to that of a 0.5
McFarland standard.
2. Inoculate a Mueller‑Hinton agar plate with this
suspension in accordance with CLSI M100‑S16 (M2)
guidelines for disc diffusion testing.
3. Place the amoxicillin‑ clavulanic acid disc towards the
centre of the plate.
4. Carefully measure 15 mm out from the edge of that
disc at 90o angles marking the plate.
5. Place a ceftazidime disk on the plate so that its inner
edge is 15 mm (the mark) from the amoxicillin‑
clavulanic acid disc (See Figure 1 KB-ESBL Template
or Figure 2 IC ESBL Screen).
Procedure cont.
6. Do the same with cefotaxime (or ceftriaxone), aztreonam
and cefpodoxime discs so that they are spaced 90o apart
and 15mm from the centre disc.
7. Place one each of cefoxitin, ceduroxime, cefepime and
pipercillin/tazobactum discs in any available space
remaining on the plate.
8. Incubate 35oC, in O2 x 18-24 hours and record the zone
diameters for the all cephalosporins as per CLSI guidelines.
9. For E. coli, Klebsiella species and Proteus species, instead
of using standard cut offs to determine S, I or R, screening
test cut offs are used and interpretations as R and S are
reported if zone size is < or > of these screening
breakpoints.
 Figure 1. KB -ESB L Templ ate

FOX CPD

15 mm
AMC

CRO 15 mm 15 mm CAZ

15 mm

ATM

TZP

CXM FEP

Placement of antibiotics for ESBLS testing

 Figure 2. IC-ESBL Template
To be used for Infection Control ESBL Screen isolates
Interpretation

Note:
The following applies to cefpodoxime-nonsusceptible E. coli, Klebsiella
species and Proteus species only.
1. After incubation, measure the diameters of the zone of complete
inhibition with calipers/ruler. Measure at the narrowest side of the
zone.
2. Document zone size for all antibiotics into the LIS.
3. Observe for potentiation of the inhibition zone (i.e. increase in the
inhibition zone) of any one of Cefpodoxime, Ceftazidime, ceftriaxone or
aztreonam when combined with clavulanic acid (enter Yes or No to the
“drug” named “Potentiation” in the LIS).
4. If a reduction of zone of inhibition of any one of Cefpodoxime,
Ceftazidime, ceftriaxone or aztreonam when combined with clavulanic
acid is observed (i.e. a D zone formation), enter Yes or No to the
“drug” named “D zone” in the LIS. Recheck the identification of the
isolate and repeat testing if the identification is questionable.
NCCLS INTERPRETATION FOR ESBLS
The National Committee for Clinical Laboratory Standards (NCCLS) has
developed broth microdilution and disk diffusion screening tests using
selected antimicrobial agents (1). Each Klebsiella pneumoniae, K. oxytoca,
or Escherichia coli isolate should be considered a potential ESBL-producer
if the test results are as follows:
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
ceftriaxone < 25 mm ceftriaxone > 2 µg/ml
The sensitivity of screening for ESBLs in enteric organisms can vary
depending on which antimicrobial agents are tested. The use of more than
one of the five antimicrobial agents suggested for screening will improve
the sensitivity of detection. Cefpodoxime and ceftazidime show the highest
sensitivity for ESBL detection.
Class A ESBL present

I. Potentiation of the inhibition zone of any

one of Cefpodoxime, Ceftazidime,
ceftriaxone or Aztreonam when combined
with clavulanic acid (see below for
examples of different patterns of
potentiation that can be seen with
organisms that contain Class A ESBLs)
II. Susceptibility to cefoxitin.
III. Susceptibility or resistance to any one of
Ceftazidime, ceftriaxone or Aztreonam
 FOX

ATM

AMC

CAZ

CRO
CPD
Detection of ESBLS
Detection of ESBLS
Class A and Class C ESBL present

I. Potentiation of the inhibition zone of any

one of Cefpodoxime, Ceftazidime,
ceftriaxone or Aztreonam when
combined with clavulanic acid
II. Resistant or Intermediate to cefoxitin.
III. Susceptibility or resistance to any one
of Ceftazidime, ceftriaxone or
Aztreonam
Class C‑ESBL present
I. No potentiation with clavulanic acid
II. Resistance or Intermediate to cefoxitin
III. Resistance to any one of ceftazidime,
ceftriaxone or Aztreonam.
Inducible Class C‑ESBL present
I. No potentiation with clavulanic acid
II. Resistance or Intermediate to cefoxitin
III. Susceptible, Intermediate or Resistance
to any one of Ceftazidime, ceftriaxone
or Aztreonam.
IV. D zone with clavulanic acid
ESBL not Class A or Class C present
I. No potentiation with clavulanic acid
II. Susceptibility to cefoxitin
III. Resistance to any one of ceftazidime,
ceftriaxone or Aztreonam
ESBL absent
I. No potentiation with clavulanic acid
II. Susceptibility or resistance to cefoxitin
III. Susceptibility to all of Ceftazidime,
ceftriaxone or Aztreonam
 Double disc diffusion and Etest to
detect ESBLS
• Double disc diffusion

• Etest
Technique type Test Advantages Disadvantages

Clinical Standard NCCLS Easy to use, performed in every ESBLs not always “resistant”
microbiology interpretive lab
criteria

NCCLS ESBL Easy to use and interpret Sensitivity depends on choice of oxyimino-cephalosporin
confirmatory test

Double-disk Easy to use, easy to interpret Distance of disk placement for optimal sensitivity not
approximation standardized
test

Three-dimensional test Sensitive, easy to interpret Not specific for ESBLS, labor intensive

Etest ESBL strips Easy to use Not always easy to interpret, not as sensitive as double-disk test

Vitek ESBL test Easy to use, easy to interpret Reduced sensitivity

Molecular DNA probes Specific for gene family (e.g., Labor intensive, cannot distinguish between ESBLs and non-
detection TEM or SHV) ESBLs, cannot distinguish between variants of TEM or SHV

PCR Easy to perform, specific for gene Cannot distinguish between ESBLs and non-ESBLs, cannot
family (e.g., TEM or SHV) distinguish between variants of TEM or SHV

Oligotyping Detects specific TEM variants Requires specific oligonucleotide probes, labor intensive, cannot
detect new variants

PCR-RFLP Easy to perform, can detect Nucleotide changes must result in altered restriction site for
specific nucleotide changes detection

PCR-SSCP Can distinguish between a Requires special electrophoresis conditions

number of SHV variants

LCR Can distinguish between a Requires a large number of oligonucleotide primers

number of SHV variants

Nucleotide The gold standard, can detect all Labor intensive, can be technically challenging, can be difficult
sequencing variants to interpret manual methods