Extended spectrum β-lactamases (ESBLs) pose challenges for detection and treatment. ESBLs hydrolyze many penicillins and cephalosporins but are inhibited by β-lactamase inhibitors. Delayed or incorrect detection of ESBL producers can lead to inappropriate cephalosporin treatment and worse outcomes. Laboratory detection of ESBLs is complex, requiring screening methods like combination disks or double disk synergy tests followed by confirmatory tests. Genotypic methods can also detect ESBL genes. Accurate detection is important for infection control and antibiotic stewardship given ESBL producers' resistance and transmission risks.

1. Extended Spectrum
β-Lactamases:
Challenges in Laboratory
Detection and Implications on
Therapy
Dr. Iman M. Fawzy
Clinical Pathology MD, PhD
Mansoura, Egypt

2. ESBL
Extended spectrum β-lactamase (ESBL)-producing
organisms pose unique challenges to
clinical microbiologists,
clinicians,
infection control professionals and
antibacterial-discovery scientists.

3. 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.

4. β-lactam antibiotics
– Penicillin
– Cephalosporin
– Monobactam
– Carbapenem

5. 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

6. 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)

7. 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 .

8. 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.

9. 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.

10. 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)

11. Major groups of -lactamases
Functi Major Molec
onal subgr ular
group oup
class
1
2
Functional group
Inhibit
ion by
clavula
nate
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-
+

12. Major groups of -lactamases
Functi Major Molec
onal subgr ular
group oup
class
2
Functional group
Inhibit
ion by
clavula
nate
Cloxacillin- (oxacillin)- hydrolyzing
enzymes
A
Cephalosporinases, confer resistance
to monobactams
+
2f
4
D
2e
3
2d
+/-
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
-

13. Selected -lactamases of gram-negative bacteria
lactam
ase
Examples
Broad- TEM- , TEM- ,
spectru SHVm
OXA family
Extend TEM family, SHV
edfamily
spectru
m
Substrates
Penicillin G,
aminopenicillins,
carboxypenicillins,
piperacillin, narrowspectrum
cephalosporins
Broad-spectrum
group plus
cloxacillin,
methicillin, and
oxacillin
Inhibiti
on by
clavula
nate*
Ambler’s
class /
Bush’s
class
+++
A / 2b
+
D / 2d
Broad-spectrum
++++
A / 2be
group plus oxyiminocephalosporins, and
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.
monobactam

14. Selected -lactamases of gram-negative bacteria
lactamas
e
Examples
Substrates
Inhibiti Ambler’
on by s class/
clavula Bush’s
nate*
class
AmpC
ACC- , ACT- , CFE-
Expandedspectrum group
CMY family, DHA- , plus
FOX
cephamycins
family, LAT family,
MIRMOX- , MOX-
0
C/ 1
Carbapen
emase
IMP family, VIM
family,
GIM- , SPM(metallo-enzymes)
0
B/3
+++
A / 2f
Expandedspectrum group
plus
cephamycins and
carbapenems
KPC- , KPC- , KPC- Same as for IMP
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.

15. Common ESBL producers
• Klebsiella
pneumoniae
• Escherichia coli
• Proteus mirabilis
• Enterobacter cloacae
• Non-typhoidal
Salmonella (in some
countries)
15

16. 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

17. 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)

18. Survival of the fittest
Resistant bacteria survive, susceptible ones die
Mutant emerges
slowly
Sensitive cells
killed by antibiotic
Mutant’s progeny
overrun

19. The Fight
PG
O
N
cell
LYSIS

20. The Fight
PG
β-lactamase
O
N
cell

21. The Fight
PG
β-lactamase
O
N
Inhibitor
cell

22. The Fight
PG
β-lactamase
Inhibitor
O
N
cell
LYSIS

23. Sites of infection
Intra abdominal
infections
6%
Others
5%
Pneumonia
11%
Skin and soft
tissues
12%
Bactremia
11%
UTI
55%

24. Laboratory Detection of ESBL
Phenotypic
Methods
Screening
methods
Genotypic
Methods
Confirmatory
Methods

25. CLSI 2013

26. CLSI 2013

27. 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

28. CLSI 2013

29. CLSI 2013

30. 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

32. Difference > 5 mm
Cefotaxim
Ceftazidim
Cefotaxim +
Clav
Ciftazidim +
Clav

33. Difference > 5 mm
Difference > 5 mm

36. 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)

37. Ceftriaxone
Amox-clav
Ceftazidime
Azteonam
Cefotaxime

40. Ceftazidim
Augmentin
Cefotaxim

42. Augmentin
Ceftazidime
Cefotaxime

44. Ceftriaxone
Cefotaxime
Augmentin

45. AMC
AMC
AMC

46. 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.

47. 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.

48. 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

49. 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)

50. Cefotaxime
Cefotaxime
+
clavulanate

51. MIC =16
Ceftaz
MIC= 0.25
Ceftaz/CA

53. 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

54. 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

55. Other confirmatory methods
6- Automated instruments
• Measure MICs and compare the growth of bacteria in
presence of cephalosporin vs. cephalosporin clavulanic acid

56. Vitek ESBL
confirmatory test
Phoenix ESBL test
(BD)
Microscan
ESBL Panel

57. 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

58. Control strains

59. 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

60. 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

62. 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.

63. 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.

64. ESBL+ AmpC
Amox-Clav
Cefepime

65. ESBL+ AmpC

66. ESBL+ AmpC
Cefotaxime
Cefipime
Cefoxitin
Augmentin
Cefpodoxime +
Clavulanic
Ceftazidime
Cefpodoxime

67. ESBL+ AmpC
ESBL and AmpC
ESBL positive
clavulanate
enhancement present
AmpC positive
cefepime: S
cefoxitin: R

68. ESBL+ AmpC
AmpC
Fox: R
Clav: R
ESBL
Zone
enhancement

69. AmpC
AmpC
cefepime : S
cefoxitin : R
no clavulanate
enhancement=
ESBL negative

70. ESBL+ Carbapenemase
ESBL +
carbapenemases
ESBL positive
clavulanate
enhancement
present
carbapenemase
production
resistance to
carbapenem agents

71. 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.

72. • Two antibiograms of ESBL producing strain. Note the
difference in zones and synergistic effect around the
amoxicillin-clavulanate pills due to different
inoculum concentration.

74. 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.

75. 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.

76. -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.

77. 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)

78. ESBL are Emerging Challenges
•
•
•
•
•
•
•
multiple enzymes
High-Risk clones
globally disseminated
hospital, community acquired
High rates
Challenge of intestinal carriage
extra-human reservoirs

79. 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

80. Prevention
– ICU is hot spot
– Hands of healthcare workers, family, visitors
– thermometer
– Ultrasound gel
– Flag records
– Education
– Contact precautions
– Transfer between wards & hospitals

81. Still the best way to prevent spread of
infections and drug resistance is ……

82. 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

83. 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

84. 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 .

85. 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.

86. 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.

87. THANK YOU