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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 intraabdominal 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-βlactam 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
Functional Major Molecular
group
subgroup
class
1
2
Functional group
Inhibition by
clavulanate
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)
-
2a
A
Penicillinases, confer resistance to all penicillins, primarily from
Staphylococcus and enterococci
+
2b
A
Broad-spectrum -lactamases (penicillinases/cephalosporinases) ,
primarily from GNB.
+
2be
A
ESBLs, confer resistance to oxyimino-cephalosporins and
monobactams.
+
2br
A
Inhibitor-resistant TEM (IRT) -lactamases
2c
A
Carbenicillin-hydrolyzing enzymes
- (+ for
tazobactam)
+
Major groups of -lactamases
Functional Major Molecular
group
subgroup
class
2
3
4
Functional group
Inhibition by
clavulanate
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)
+
3a, 3b, 3c
B
Metallo--lactamases (zinc based carbapenemases), confer
resistance to carbapenems and all -lactam classes, except
monobactams.
-
Miscellaneous unsequenced enzymes that do not fit into other
groups
-
Functional group classified by Bush-Jacoby-Medeiros.
Molecular group classified by Ambler.
Selected -lactamases of gram-negative bacteria
-lactamase
Broadspectrum
Extendedspectrum
Examples
Substrates
Inhibition by Ambler’s class /
clavulanate* Bush’s class
TEM-1, TEM-2, SHV-1
Penicillin G, aminopenicillins,
carboxypenicillins, piperacillin,
narrow-spectrum cephalosporins
+++
A / 2b
OXA family
Broad-spectrum group plus
cloxacillin, methicillin, and oxacillin
+
D / 2d
TEM family, SHV family
Broad-spectrum group plus
oxyimino-cephalosporins, and
monobactam (aztreonam)
++++
A / 2be
CTX-M family
Expanded-spectrum group plus, for
some enzymes, cefepime
++++
A
OXA family
Same as for CTX-M family
+
D / 2d
++++
A
Others (PER-1, PER-2,
Same as for TEM family and SHV
family
BES-1, GES/IBC family,
SFO-1, TLA-1, VEB-1, VEB2)
*+, +++ , and ++++ denote relative sensitivity to inhibition.
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.
Selected -lactamases of gram-negative bacteria
-lactamase
AmpC
Examples
ACC-1, ACT-1, CFE-1,
CMY family, DHA-2, FOX
family, LAT family, MIR-1,
MOX-1, MOX-2
Substrates
Inhibition by
Ambler’s
clavulanate* class/ Bush’s
class
Expanded-spectrum group plus
cephamycins
0
C/ 1
Carbapenemase IMP family, VIM family,
Expanded-spectrum group plus
GIM-1, SPM-1 (metallo-enzymes) cephamycins and carbapenems
0
B/3
KPC-1, KPC-2, KPC-3
Same as for IMP family, VIM
family, GIM-1, and SPM-1
+++
A / 2f
OXA-23, OXA-24, OXA-25,
OXA-26, OXA-27, OXA-40,
OXA-48
Same as for IMP family, VIM
family, GIM-1, and SPM-1
+
D / 2d
*+, +++ , and ++++ denote relative sensitivity to inhibition.
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
TEM, SHV
Major sources
E. coli, K. pneumoniae
Cefotaxime hydrolyzing S. Typhimurium, E. coli, K. pneumoniae
(CTX-M)
Oxacillin hydrolyzing
(OXA)
P. aeruginosa
PER-1
PER-2
P. aeruginosa, A. baumanii, S. Typhimurium
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
slowly
Sensitive cells
killed by antibiotic
Mutant’s progeny
overrun
The Fight
PG
O
N
cell
LYSIS
The Fight
PG
β-lactamase
O
N
cell
The Fight
PG
β-lactamase
O
N
Inhibitor
cell
The Fight
PG
β-lactamase
Inhibitor
O
N
cell
LYSIS
Sites of infection
Intra abdominal
infections
6%
Others
5%
Pneumonia
11%
Skin and soft
tissues
12%
Bactremia
11%
UTI
55%
Laboratory Detection of ESBL
Phenotypic
Methods
Screening
methods
Genotypic
Methods
Confirmatory
Methods
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
Ceftaz
Cefotax/CA
Cefotax
Cefotaxime/CA
Negative ESBL
Ceftaz/CA
Ceftaz
Cefotax
Difference > 5 mm
Cefotaxim
Ceftazidim
Cefotaxim +
Clav
Ciftazidim +
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 amoxicillinclavulanic acid
– Result: Enhanced inhibition (A keyhole or ghost zone)
Ceftriaxone
Amox-clav
Ceftazidime
Azteonam
Cefotaxime
Ceftazidim
Augmentin
Cefotaxim
Augmentin
Ceftazidime
Cefotaxime
Ceftriaxone
Cefotaxime
Augmentin
AMC
AMC
AMC
30 mm distance between
discs (center to center)
20 mm distance between
discs (center to center)
AMC, amoxicillin-clavulanate; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; FEP, cefepime;
CPO, cefpirome.
30 mm distance between
discs (center to center)
20 mm distance between
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 -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 ESBLproducing 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,
ceftriaxone
R
R
S/R
S
Cefepime
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
Cefipime
Cefoxitin
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 highinoculum,
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 carbapenemresistant 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 multiresistance.
– 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 rethought 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 ESBLproducing 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