1. Dr. Iman M. Fawzy Clinical Pathology MD, PhD Mansoura, Egypt
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
nitrogen containing ring
Bacteriocidal – mechanism is that the beta-lactam ring binds to and inactivate PBPs, preventing crosslinking of peptidoglycan = weakening = lysis

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)
Intrinsic resistance = inherent or innate (not acquired) resistance, which is reflected in wildtype antimicrobial patterns of all or almost all representatives of a species.
Intrinsic resistance is so common that susceptibility testing is unnecessary.
• Acquired resistance = antimicrobial resistance
in a bacterium that was previously susceptible
that occurs as a result of:
– Chance gene mutation
– Acquisition of R genes from another bacterium

7. 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 .

8. 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.

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
Functional group
Major subgroup
Molecular class
Inhibition by clavulanate 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
Broad-spectrum -lactamases (penicillinases/cephalosporinases) , primarily from GNB.
2be
ESBLs, confer resistance to oxyimino-cephalosporins and monobactams.
2br
Inhibitor-resistant TEM (IRT) -lactamases
- (+ for tazobactam) 2c
Carbenicillin-hydrolyzing enzymes
Functional classification (Bush-Jacoby-Medeiros: 1-4) – spectrum of ATB substrate profile, enzyme inhibitor profile, enzyme net charge, hydrolysis rate, binding affinity, isoelectric focusing, protein molecular weight, amino acid composition
Molecular classification (Ambler’s classification: A-D) – nucleotide and amino acid sequence

12. Major groups of -lactamases
Functional group
Major subgroup
Molecular class
Inhibition by clavulanate 2 2d D
Cloxacillin- (oxacillin)- hydrolyzing enzymes
+/- 2e A
Cephalosporinases, confer resistance to monobactams + 2f
Carbapenem-hydrolyzing enzymes with active site serine (serine based carbapenemases) 3
3a, 3b, 3c B
Metallo--lactamases (zinc based carbapenemases), confer resistance to carbapenems and all -lactam classes, except monobactams. - 4
Miscellaneous unsequenced enzymes that do not fit into other groups
Functional group classified by Bush-Jacoby-Medeiros.
Molecular group classified by Ambler.
Functional classification (Bush-Jacoby-Medeiros: 1-4) – spectrum of ATB substrate profile, enzyme inhibitor profile, enzyme net charge, hydrolysis rate, binding affinity, isoelectric focusing, protein molecular weight, amino acid composition
Molecular classification (Ambler’s classification: A-D) – nucleotide and amino acid sequence

13. Selected -lactamases of gram-negative bacteria
Examples
Substrates
Inhibition by clavulanate*
Ambler’s class / Bush’s class
Broad-spectrum
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
Extended-spectrum
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
Same as for CTX-M family
Others (PER-1, PER-2,
BES-1, GES/IBC family,
SFO-1, TLA-1, VEB-1, VEB2)
Same as for TEM family and SHV family
*+, +++ , and ++++ denote relative sensitivity to inhibition.
Peterson DL. Am J Med 2006; 119 (6 Suppl 1):S20-8.

14. Selected -lactamases of gram-negative bacteria
Examples
Substrates
Inhibition by clavulanate*
Ambler’s class/ Bush’s class
AmpC
ACC-1, ACT-1, CFE-1,
CMY family, DHA-2, FOX
family, LAT family, MIR-1,
MOX-1, MOX-2
Expanded-spectrum group plus
cephamycins
C / 1
Carbapenemase
IMP family, VIM family,
GIM-1, SPM-1 (metallo-enzymes)
cephamycins and carbapenems
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 +
D / 2d
*+, +++ , and ++++ denote relative sensitivity to inhibition.
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)

16. Common ESBL producers Type Major sources TEM, SHV
E. coli, K. pneumoniae
Cefotaxime hydrolyzing (CTX-M)
S. Typhimurium, E. coli, K. pneumoniae
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
Antibiotics do not cause mutations!
Antibiotics can select for the emergence and spread of resistance determinants by two different mechanisms.
First, they can select for the appearance of the resistance determinant in a pathogenic species. If resistance emerges due to mutation of a cellular gene, the presence of an antibiotic in a significant concentration in the environment will provide selective advantage for survival of the resistant mutant.
Second, the presence of selective concentrations of antibiotics can promote the spread of resistant strains from patient to patient by creating an environment that favours establishment of colonization with these strains. If the human gastrointestinal tract is though of as an intensely competitive environment in which many different species of bacteria compete for a limited number of resources, the presence of a resistance determinant in a given species can be a benefit in that the the level of competition for resources (normally created by the presence of competitors susceptible to the antibiotic) is reduced. In this setting, bacteria that would normally be poor competitors for limited resources can survive and even thrive.
Mutant’s progeny
overrun
Mutant emerges
slowly
Sensitive cells
killed by antibiotic

19. The Fight PG
cell N O
LYSIS

20. The Fight PG
β-lactamase
cell N O

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

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

23. Sites of infection

24. Laboratory Detection of ESBL
Phenotypic Methods
Screening methods
Confirmatory Methods
Genotypic 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
Cefotax/CA
Ceftaz
Cefotax
Cefotaxime/CA
Negative ESBL
Ceftaz/CA
Ceftaz
Cefotax
jhindler nltn MW tele #

32. Difference > 5 mm
Ceftazidim
Cefotaxim
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 amoxicillin-clavulanic acid
Result: Enhanced inhibition (A keyhole or ghost zone)

37. Ceftriaxone
Cefotaxime
Amox-clav
Ceftazidime
Azteonam

40. Augmentin
Cefotaxim
Ceftazidim

42. Augmentin
Ceftazidime
Cefotaxime

44. Ceftriaxone
Cefotaxime
Augmentin

45. AMC
AMC
AMC

46. 30 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)
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
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. Ceftaz
MIC =16
MIC= 0.25
Ceftaz/CA
jhindler nltn MW tele #

53. 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

54. 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

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 S R Cefoxitin, cefotetan
Inhibitors (pip/tazo, amp/sulbactam, amox/clav) S R
Cefoxitin, cefotetan
Ceftazidime, ceftriaxone
Cefepime
S/R

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 Cefoxitin Cefipime Augmentin
Cefpodoxime + Clavulanic
Cefpodoxime
Ceftazidime

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 high-inoculum, physicians should avoid cephalosporins if risk of ESBL-producing organism is suspected.

72. Two antibiograms of ESBL producing strain
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 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.

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 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

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 Institutional 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 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 .

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 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.

87. THANK YOU