Extended Spectrum

β-Lactamases:
Challenges in Laboratory
Detection and Implications on
Therapy
Dr. Iman M. Fawzy
Clinical Pathology MD, PhD
Mansoura, Egypt
 ESBL
Extended spectrum β-lactamase (ESBL)-producing
organisms pose unique challenges to

clinical microbiologists,
clinicians,
infection control professionals and
antibacterial-discovery scientists.
 Why we need esbl detection?
• ESBL-producing Enterobacteriaceae have been
responsible for numerous outbreaks of infection
throughout the world
• ESBL pose challenging infection control issues.
• ESBLs are clinically significant and indicate the
appropriate antibacterial agents.
• Unfortunately, the laboratory detection of ESBLs
can be complex and, at times, misleading.
 β-lactam antibiotics

– Penicillin
– Cephalosporin
– Monobactam
– Carbapenem
 β lactamases
Beta lactamases are enzymes produced by some
bacteria that hydrolyze beta lactam antibiotics

– Penicillinases, Cephalosporinases
– Extended spectrum β-lactamases (ESBL)
– Metallo β lactamases
– Amp C
– Carbapenemase
 Definition of ESBL
• ESBLs are enzymes

– hydrolyzing most penicillins and cephalosporins,

and monobactam (aztreonam).

– but not cephamycins and carbapenems

– Susciptable to β-lactamase inhibitors

(clavulanate, sulbactam and tazobactam)
 Clinical significance
• ESBLs destroy cephalosporins, main hospital
antibiotics, given as first-line agents to many
severely-ill patients, including those with intra-
abdominal infections, community-acquired
pneumonias and bacteraemias.

• Delayed recognition inappropriate treatment of

severe infections caused by ESBL producers with
cephalosporins  ↑↑mortality .
 Clinical significance
• ESBL-mediated resistance is not always obvious to
all cephalosporins in vitro.

• Many ESBL producers are multi-resistant to non-β-

lactam antibiotics such as quinolones,
aminoglycosides and trimethoprim, narrowing
treatment options.
 Spread

• direct and indirect contact

• with colonized/infected patients and

• contaminated environmental surfaces.

• ESBLs are most commonly spread via unwashed

hands of health care providers.
 Risk factors
• Critically ill patients, Immunosuppression
• Prolonged hospital or ICU unit stay
• Invasive procedures: intubation, mechanical
ventillation, catheter
• Long-term dialysis within 30 days
• Family member with multidrug-resistant pathogens
• Prior antibiotic use in last 3 months
• High frequency of antibiotic resistance in the
community or in the specific hospital unit
• Patient who previously had an antibiotic-resistant
organism (e.g., MRSA, VRE)
 Major groups of -lactamases
Func Inhibi
Majo
tiona Mole tion
r
l cular Functional group by
subg
grou class clavul
roup
p anate
1 C Cephalosporinases, often -
chromosomal enzymes in GNB but
may be plasmid-encoded, confer
resistance to all classes of -
lactams, except carbapenems
(unless combine with porin
change)
2 2a A Penicillinases, confer resistance +
to all penicillins, primarily from
Staphylococcus and enterococci
2b A Broad-spectrum -lactamases +
(penicillinases/cephalosporinases)
, primarily from GNB.
 Major groups of -lactamases
Func Inhibi
Majo
tiona Mole tion
r
l cular Functional group by
subg
grou class clavul
roup
p anate
2 2d D Cloxacillin- (oxacillin)- hydrolyzing +/-
enzymes
2e A Cephalosporinases, confer +
resistance to monobactams
2f A Carbapenem-hydrolyzing enzymes +
with active site serine (serine
based carbapenemases)
3 3a, B Metallo--lactamases (zinc based -
3b, carbapenemases), confer
3c resistance to carbapenems and all
-lactam classes, except
monobactams.
 Selected -lactamases of gram-negative bacteria
- Examples Substrates Inhibit Ambler’
lactam ion by s class /
ase clavul Bush’s
anate* class
Broad- TEM-1, TEM-2, Penicillin G, +++ A / 2b
spectr SHV-1 aminopenicillins,
um carboxypenicillins,
piperacillin,
narrow-spectrum
cephalosporins
OXA family Broad-spectrum + D / 2d
group plus
cloxacillin,
methicillin, and
oxacillin
Extend TEM family, Broad-spectrum ++++ A / 2be
ed- SHV family group plus
spectr oxyimino-
um cephalosporins,
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.
 Selected -lactamases of gram-negative bacteria
- Examples Substrates Inhibit Amble
lactamas ion by r’s
e clavul class/
anate* Bush’s
class
AmpC ACC-1, ACT-1, Expanded- 0 C/ 1
CFE-1, spectrum group
CMY family, DHA- plus
2, FOX cephamycins
family, LAT
family, MIR-1,
MOX-1, MOX-2
Carbape IMP family, VIM Expanded- 0 B/3
nemase family, spectrum group
GIM-1, SPM-1 plus
(metallo- cephamycins
enzymes) and
carbapenems
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.
 Common ESBL producers
• Klebsiella
pneumoniae
• Escherichia coli
• Proteus mirabilis
• Enterobacter cloacae
• Non-typhoidal
Salmonella (in some
countries)

15
 Common ESBL producers
Type Major sources

TEM, SHV E. coli, K. pneumoniae

Cefotaxime hydrolyzing S. Typhimurium, E. coli, K. pneumoniae

(CTX-M)
Oxacillin hydrolyzing P. aeruginosa
(OXA)
PER-1 P. aeruginosa, A. baumanii, S. Typhimurium
PER-2 S. Typhimurium
VEB-1 E. coli, P. aeruginosa
 Mechanisms of resistance
• The majority of ESBLs are acquired enzymes,
encoded by plasmids.

• Different resistance phenotypes to:

– Different expression levels
– Different biochemical characteristics such as
activity against specific β-lactams
– co-presence of other resistance mechanisms
(other β-lactamases, efflux, altered permeability)
 Survival of the fittest
Resistant bacteria survive, susceptible ones die

Mutant emerges Sensitive cells Mutant’s progeny

slowly killed by antibiotic overrun
 The Fight

PG

N cell
O
LYSIS
 The Fight

PG

β-lactamase

N cell
O
 The Fight

PG

β-lactamase

N Inhibitor cell
O
 The Fight

PG

β-lactamase

Inhibitor

N cell
O
LYSIS
 Sites of infection

In-
tra Others
ab- 5%
do
mi-
nal
in-
fec-
tion
s
6%
Pne
Skin um
and UTI
oni 55%
soft a
tis- 11%
sue
s
12% Bactremia
11%
Laboratory Detection of ESBL
Phenotypic Genotypic
Methods
Screeni Methods
ng
metho
ds
CLSI 2013
CLSI 2013
 Confirmatory methods
• 1- Combination disk
– Uses 2 disks of 3rd cephalosporin alone and
combined with clavulanic acid
– An increase of ≥5 mm in zone inhibition with use
of the combination disk
Disc with cephalosporin
+ clavulanic acid

Disc with
cephalosporin
alone
CLSI 2013
CLSI 2013
 Positive ESBL Cefotaxime/CA
Ceftaz

Cefotax
Ceftaz/CA

Ceftaz/CA Difference > 5 mm

Positive ESBL
Cefotax/CA

Ceftaz Cefotax

Cefotaxime/CA
Negative ESBL

Ceftaz/CA
Ceftaz
Cefotax
 Difference > 5 mm

Cefotaxim Ceftazidim

Cefotaxim + Ciftazidim +
Clav Clav
 Difference > 5 mm

Difference > 5 mm
 Phenotypic conformation
2- Double disk approximation or double disk
synergy
– Disk of 3rd cephalosporin placed 30 mm from amoxicillin-
clavulanic acid

– Result: Enhanced inhibition (A keyhole or ghost zone)

 Ceftriaxone

Cefotaxime
Amox-clav
Ceftazidime

Azteonam
 Augmentin Cefotaxim
Ceftazidim
 Augmentin
Ceftazidime
Cefotaxime
 Ceftriaxone

Cefotaxime
Augmentin
AMC AMC AMC
 30 mm distance between 20 mm distance between
discs (center to center) discs (center to center)

AMC, amoxicillin-clavulanate; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; FEP, cefepime;
CPO, cefpirome.
 30 mm distance between 20 mm distance between
discs (center to center) discs (center to center)

AMC, amoxicillin-clavulanate; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; FEP, cefepime;
CPO, cefpirome.
 Phenotypic conformation
• 3- Broth Microdilution
MIC of 3rd cephalosporin alone and combined with clavulanic acid
>3-two fold serial dilution decrease in MIC of either cephalosporin in the
presence of clavulanic acid compared to its MIC when tested alone.
Ceftazidim MIC =8 μg/mL
Ceftazidime + Clavulanate= 1 μg/mL
Or MIC ratio≥8

4- MIC broth dilution

MIC of 3rd cephalosporin alone and combined with clavulanic acid
MIC of 3rd cephalosporin alone and combined with clavulanic acid
A decrease in the MIC of the combination of > 3-two fold dilutions
 Phenotypic conformation
• 5- E-test (MIC ESBL strips)
• Two-sided strip containing cephalosporin on one side
and cephalosporin -clavulanic acid on the other
• MIC ratio ≥8
•>8 fold reduction in MIC in presence of CA= ESBL
• or Phantom zone (deformed ellipse)
Cefotaxime

Cefotaxime
+
clavulanate
 MIC =16
Ceftaz

MIC= 0.25

Ceftaz/CA
 Other confirmatory methods
Cica b-Test
uses the chromogenic cephalosporin HMRZ-86,4,5 + inhibitors to determine rapidly
whether an isolate has a metallo-β-lactamase (MBL), ESBL, or hyperproduced AmpC
enzyme , a control strip with no inhibitor, to detect hydrolysis of extended-spectrum
cephalosporins

No inhibitor

Mercaptoacetic acid to inhibit MBL

Clavulanate to inhibit ESBL

Boronic acid to inhibit AmpC

 Other confirmatory methods
Brilliance ESBL agar
• identification of ESBL-
producing E. coli, Klebsiella,
Enterobacter, Serratia and
Citrobacter group, directly
from clinical samples.
• two chromogens that
specifically target enzymes
green and blue colonies
• Negative pink.
• Proteus, Morganella and
Providencia  tan-coloured
colonies with a brown halo
 Other confirmatory methods

6- Automated instruments
• Measure MICs and compare the growth of bacteria in
presence of cephalosporin vs. cephalosporin -
clavulanic acid
 Vitek ESBL
confirmatory test Phoenix ESBL test (BD)

Microscan
ESBL Panel
 Genotypic confirmation
• Molecular detection
– PCR
– RFLP
– gene sequencing
– DNA microarray-based method

• Targets specific nucleotide sequences to detect

different variants of TEM and SHV genes
Control strains
 Pitfalls in ESBL tests
AmpC β-lactamases
• third-generation cephalosporins: resistance ,
• cephamycins, e.g. a cefoxitin: resistance
• Cefepime: sensitive.

Carbapenemases
The presence of ESBLs may also be masked by
carbapenemases
 ESBLs vs AmpCs

ESBLs AmpCs

Inhibitors (pip/tazo,
amp/sulbactam, amox/clav) S R

Cefoxitin, cefotetan S R
Ceftazidime, R R
ceftriaxone
Cefepime S/R S
 Pitfalls in ESBL tests
ESBL+ AmpC β -lactamases:
• Especially in Enterobacter spp., Citrobacter,
Morganella, Providencia and Serratia.
• The AmpC enzymes may be induced by
clavulanate (which inhibits them poorly) and
may then attack the cephalosporin, masking
synergy arising from inhibition of the ESBL.
 Pitfalls in ESBL tests
• Screening criterion for ESBL presence among
AmpC‑producing Enterobacter, C. freundii and
Serratia is Cefepime MIC > 1 ug/ml (inhibition
zone< 26 mm).
• Use of Cefepime is more reliable to detect
these strains because high AmpC production
has little effect on cefepime activity.
ESBL+ AmpC

Amox-Clav
Cefepime
ESBL+ AmpC
ESBL+ AmpC
Cefotaxime Cefoxitin

Cefipime Augmentin

Cefpodoxime +
Clavulanic

Ceftazidime Cefpodoxime
ESBL+ AmpC

ESBL and AmpC

ESBL positive
clavulanate
enhancement present

AmpC positive
cefepime: S
cefoxitin: R
ESBL+ AmpC
AmpC
Fox: R
Clav: R

ESBL
Zone
enhancement
AmpC

AmpC
cefepime : S
cefoxitin : R

no clavulanate
enhancement=
ESBL negative
ESBL+ Carbapenemase

ESBL +
carbapenemases

ESBL positive
clavulanate
enhancement
present

carbapenemase
production
resistance to
carbapenem agents
 ESBLs and the inoculum effect
• In vitro: the MICs of cephalosporins rise as the
inoculum of ESBL- producing organisms
increases.
• In vivo: Intra-abdominal abscesses and
pneumonia are some of the clinical settings
where organisms are present in high-
inoculum, physicians should avoid
cephalosporins if risk of ESBL-producing
organism is suspected.
• Two antibiograms of ESBL producing strain. Note the
difference in zones and synergistic effect around the
amoxicillin-clavulanate pills due to different inoculum
concentration.
 Reporting
If ESBL:
Resistant, for all penicillins, cephalosporins, and
monobactams

Report beta lactam inhibitor drugs as they test.

If ESBL is not detected, report drugs as tested.

 Treatment
Carbapenems are the drugs of choice.

Unfortunately, use of carbapenems has been

associated with the emergence of carbapenem-
resistant bacterial species

It may be advisable to use non carbapenem

antimicrobials as the first line treatment in the less
severe infections with ESBL producing strains.
 -lactam/-lactamase inhibitor on
treatment of ESBL-producing organisms
• Most ESBLs are susceptible to clavulanate and
tazobactam in vitro,
• nevertheless some ESBL producers are resistant to
 -lactamase inhibitor due to
– Hyperproduction of the ESBLs → overwhelm inhibitor
– Co-production of inhibitor-resistant penicillinases or
AmpC enzyme
– Relative impermeability of the host strain
•  -lactam/ -lactamase inhibitor should not be used
to treat serious infections with ESBL-producing
organisms.
Summary of cephamycins on treatment
of ESBL-producing organisms
• Limited clinical data
• Generally effective against Enterobacteriaceae
producing TEM-, SHV-, and CTX-M-derived ESBLs
• Reports of cephamycins resistance development
during prolonged therapy
– Loss of outer membrane porin (porin deficient
mutant)
– Acquisition of plasmid-mediated AmpC -
lactamase (ACT-1)
 ESBL are Emerging Challenges
• multiple enzymes
• High-Risk clones
• globally disseminated
• hospital, community acquired
• High rates
• Challenge of intestinal carriage
• extra-human reservoirs
 ESBL are more complex
• Antibacterial choice is often complicated by multi-
resistance.
– Many ESBL producing organisms also express
AmpC β-lactamases
– may be co-transferred with plasmids mediating
aminoglycoside resistance.
– there is an increasing association between ESBL
production and fluoroquinolone resistance
 Prevention
– ICU is hot spot
– Hands of healthcare workers, family, visitors
– thermometer
– Ultrasound gel
– Flag records
– Education
– Contact precautions
– Transfer between wards & hospitals
Still the best way to prevent spread of
infections and drug resistance is ……
 Prevention
Individual patient level
•Avoid use of cephalosporins, aztreonam
•Avoid unnecessary use of invasive devices
•Ensure good hand hygiene before and after patient-care activities
Institutional level
•Restrict use of 3rd-generation cephalosporins
•Isolation of patient
•Investigate environmental contamination
 Recommendations
• Older agents such as aminoglycosides need
reappraisal to spare the selective pressures of a
carbapenem.

• new trials of cephalosporin/β-lactamase inhibitors

can be predicted

• oral carbapenems are urgently needed

 Recommendations
• Empirical treatment strategies may need to be re-
thought where there is a significant risk.

• Use a carbapenem until the infection has been

proved NOT to involve an ESBL producer, then to step
down to a narrower- spectrum ab .
 Recommendations
• Optimize appropriate use of antimicrobials
– The right agent, dose, timing, duration, route

• Help reduce antimicrobial resistance

– The combination of effective antimicrobial
supervision and infection control has been shown
to limit the emergence and transmission of
antimicrobial-resistant bacteria

Dellit TH et al. Clin Infect Dis. 2007;44(2):159–177; . Drew RH. J Manag Care Pharm.
2009;15(2 Suppl):S18–S23; Drew RH et al. Pharmacotherapy. 2009;29(5):593–607.
 Take Home Messages
• ESBL-producing bacterial infection is an emerging
problem worldwide.
• These organisms are associated with multi-drug
resistance causing high rate of mortality and treatment
failure.
• The significant risk factors for ESBL-producing bacterial
infection are prior use of antibiotics, especially 3rd
generation cephalosporins, and critically ill or debilitated
patients.
• Need the ESBL-laboratory testing for establish the
problem.
• Carbapenems is the drug of choice for serious ESBL-
producing bacterial infection.
• Avoiding overuse or misuse of 3rd generation
cephalosporins and implementing isolation and contact
precaution to prevent and control the ESBL outbreak.
THANK YOU