Beta lactamase diagnosis extended spectrum b lactamase and metallo b lactamase screening and confirmatory test disc diffusion test broth microdilution method molecular diagnosis and CLSI guidelines

1. ESBL and MBL:
Methods of Detection
Moderator:
Dr. Anju Mahor
Assistant Professor
Microbiology Department
MGMMC Indore
Presentor:
Dr. Ananya Verma
P.G Resident
Microbiology Department
MGMMC Indore

2. INDEX
 Defination [ESBL & MBL]
 Classification
 Methods of detection
 screening tests
 confirmatory tests
 Combined disc diffusion test
 Double disc synergy test
 E test
 Microbroth dilution test
 Molecular assay

3. What is ESBL & MBL ?

4. ESBL- Extended spectrum β
lactamases
ESBLs are enzymes that mediate resistance to
extended spectrum (third generation)
cephalosporins and Monobactams but do not
affect cephamycins or carbapenems.

5. MBL-Metallo-β-Lactamases
MBLs are a unique group of β-lactamases that
have a zinc ion at the active site. These
metalloenzymes hydrolyze carbapenems but
have poor ability to hydrolyze monobactams.
They are not inhibited by tazobactam or
clavulanic acid, but are readily inhibited by
EDTA, dipicolinic acid or 1,10-o-phenanthroline.

6. Methods of Detection
 Screening Test
1.Disk diffusion Test
2.Broth dilution method
 Confirmatory Test
1.Double Disk Synergy Test
2.Combined Disk Test
3.Broth Microdilution method
4.Etest
5.Vitek 2
6.PCR

7. Screening Test for ESBL
Production
The Clinical and Laboratories Standard Institute
(CLSI) as well as European Committee on
Antimicrobial Susceptibility testing (EUCAST)
recommended a two-step phenotypic approach
in detecting ESBL production followed by
confirmatory tests. The initial screening may be
carried out by broth microdilution or disk
diffusion method while the confirmatory test
mainly relies on the action of beta lactamase
inhibitors to enhance the zone of inhibition.

8. Test Criteria for Performance of ESBL Test
Test method Disk diffusion Broth microdilution
Medium MHA CAMHB
Antimicrobial
concentration
For K. pneumoniae,K.
oxytoca, and E. coli:
Cefpodoxime 10 µg or
Ceftazidime 30 µg or
Aztreonam 30 µg or
Cefotaxime 30 µg or
Ceftriaxone 30 µg
For P. mirabilis:
Cefpodoxime 10 µg or
Ceftazidime 30 µg or
Cefotaxime 30 µg
(Testing more than one
antimicrobial agent improves
the sensitivity of ESBL
detection.)
For K. pneumoniae,K. oxytoca, and E. Coli:
Cefpodoxime 4 µg/mL or
Ceftazidime 1 µg/mL or
Aztreonam 1 µg/mL or
Cefotaxime 1 µg/mL or
Ceftriaxone 1 µg/mL
For P. mirabilis:
Cefpodoxime 1 µg/mL or
Ceftazidime 1 µg/mL
or
Cefotaxime 1 µg/mL
Inoculum Standard disk diffusion
procedure
Standard broth dilution procedure
Incubation
conditions
35°C to 37°C; ambient air 35°C to 37°C; ambient air
Incubation
length
16–18 hours 16–18 hours

9. Test Criteria for Performance of ESBL Test
Test method Disk diffusion Broth microdilution
Results For K. pneumoniae, K.
oxytoca,and E. coli:
For K. pneumoniae, K. oxytoca,and E. coli:
Cefpodoxime zone ≤ 17 mm
Ceftazidime zone ≤ 22 mm
Aztreonam zone ≤ 27 mm
Cefotaxime zone ≤ 27 mm
Ceftriaxone zone ≤ 25 mm
For P. mirabilis:
Cefpodoxime zone ≤ 22 mm
Ceftazidime zone ≤ 22 mm
Cefotaxime zone ≤ 27 mm
Zones above may indicate
ESBL production.
Cefpodoxime MIC ≥ 8 µg/mL
Ceftazidime MIC ≥ 2 µg
Aztreonam MIC ≥ 2 µg
Cefotaxime MIC ≥ 2 µg
Ceftriaxone MIC ≥ 2 µg
For P. mirabilis:
Cefpodoxime MIC ≥ 2 µg
Ceftazidime MIC ≥ 2 µg
Cefotaxime MIC ≥ 2 µg
Growth at or above the concentrations listed
may indicate ESBL production.

10. Test ESBL Test
Test method Disk diffusion Broth microdilution
Medium MHA CAMHB
Antimicrobial
concentration
Ceftazidime 30 µg
Ceftazidime-clavulanate
30/10 µg
and
Cefotaxime 30 µg
Cefotaxime-clavulanate
30/10 µg
Ceftazidime 0.25–128 µg/mL
Ceftazidime-clavulanate 0.25/4–128/4 µg/mL
And
Cefotaxime 0.25–64 µg/mL
Cefotaxime-clavulanate 0.25/4–64/4 µg/mL
Inoculum Standard disk diffusion
procedure
Standard broth dilution procedure
Incubation
conditions
35°C to 37°C; ambient air 35°C to 37°C; ambient air
Incubation
length
16–18 hours 16–18 hours
Result A ≥ 5-mm increase in a zone
diameter for either
antimicrobial agent tested in
combination with clavulanate
vs the zone diameter of
the agent when tested alone
= ESBL
A ≥ 3 two-fold concentration decrease in an
MIC for either antimicrobial agent tested in
combination with clavulanate vs the MIC of the
agent when tested alone = ESBL

11. Disc Diffusion Method
 Principle: Antibiotic impregnated disc absorbs
moisture from the agar and antibiotic diffuses
into the agar medium. As the distance from the
disc increases,
there is logrithmic reduction in the antibiotic
concentration.
 Concomitant with the diffusion of the drug, the
bacteria that were inoculated onto the agar
surface are not inhibited by the concentration
of the antimicrobial agent continue to multiply
and there growth is visible. In areas where the
concentration of the drug is inhibitory, no growth
Antibiotic
disk
 Reference-Koneman’s color atlas and textbook of diagnostic microbiology

12. Inoculum prepration
Using a loop or swab, transfer colonies as follows.
1. Direct colony suspension method
Pick several colonies from a fresh (18- to 24-h) nonselective agar
plate to broth or 0.9% NaCl.
2. Log-phase method
a. Pick four or five isolated colonies to 3.0 to 5.0 ml of broth.
b. Incubate at 35ºC for 2 to 8 h until growth reaches the turbidity at
or above that of a 0.5 McFarland standard.
For either the log phase or direct colony suspension method,
vortex well and adjust turbidity visually with sterile broth or 0.9%
NaCl to match a 0.5 McFarland standard. Alternatively, standardize
suspension with a photometric device.

14. Inoculation of agar plate
1. Within 15 min of adjusting turbidity, dip a sterile
cotton swab into the inoculum and rotate
against the wall of the tube above the liquid to
remove excess inoculum.
2. Swab entire surface of MHA plate three times,
rotating plate approximately 60º between
streaking to ensure even distribution. Avoid
hitting the sides of the plate to avoid aerosols.
Finally, run swab around the edge of the agar to
remove any excess moisture.
3. Allow inoculated plate to stand for 3 to 15 min
before applying disks.

15. Application of disks
1. Apply disks to agar surface with dispenser or
manually with a sterile forceps.
2. Apply gentle pressure with sterile forceps or
needle to ensure complete contact of disk with
agar.
3. Do not place disks closer than 24 mm from
center to center.
a. No more than 12 disks on 150-mm plate
b. No more than 5 disks on 100-mm plate
c. Do not relocate a disk once it has made
contact with agar surface. Instead, place a new
disk in another location on the agar.

16. Antimicrobial concentration for testing
ESBL
For K. pneumoniae, K.
oxytoca, E. coli
Cefpodoxime 10 µg
Ceftazidime 30 µg
Aztreonam 30 µg
Cefotaxime 30 µg
Ceftriaxone 30 µg
For P. mirabilis
Cefpodoxime 10 µg
Ceftazidime 30 µg
Cefotaxime 30 µg
Testing more than one antimicrobial agent improves the
sensitivity of ESBL detection.
Reference- CLSI guidelines M100 table 3a

17. Incubation
1. Invert plates and incubate within 15 min of disk
application.
2. Incubate for 16 to 18 h at 35ºC in an ambient-
air incubator.

18. Results
For K. pneumoniae, K. oxytoca and
E. coli:
Cefpodoxime ≤ 17 mm
Ceftazidime ≤ 22 mm
Aztreonam ≤ 27 mm
Cefotaxime ≤ 27 mm
Ceftriaxone ≤ 25 mm
For P. mirabilis:
Cefpodoxime ≤ 22 mm
Ceftazidime ≤ 22 mm
Cefotaxime ≤ 27 mm

19. QC recommendations
E. coli ATCC 25922
Cefpodoxime 23–28 mm
Ceftazidime 25–32 mm
Aztreonam 28-36 mm
Cefotaxime 29-35 mm
Ceftriaxone 29-35 mm
K. pneumoniae ATCC 700603
Cefpodoxime 9–16 mm
Ceftazidime 10–18 mm
Aztreonam 10–16 mm
Cefotaxime 17–25 mm
Ceftriaxone 16–24 mm
reference- CLSI guidelines M100 table 3a

20. Broth Microdilution Method
PRINCIPLE:
 The broth microdilution method is used to
measure the in vitro activity of an antimicrobial
agent against a bacterial isolate.
 A sterile plastic tray containing various
concentrations of antimicrobial agents is
inoculated with a standardized number of test
bacteria. After overnight incubation at 35ºC, the
MIC is determined by observing the lowest
concentration of an antimicrobial agent
which will inhibit visible growth of the
bacterium. I.

21. Solvents and Diluents for Preparing
Stock Solutions of Antimicrobial Agent
Antimicrobial Agent Solvents Diluents
Cefpodoxime 0.10% (11.9 mmol/L)
aqueous sodium
bicarbonate
Water
Ceftazidime Sodium carbonate Water
Aztreonam Saturated solution
sodium bicarbonate
Water
Cefotaxime Water Water
Ceftriaxone Water Water
Reference-CLSI M100 ED32 Table 6A Solvents and Diluents M07

22. Stock solution prepration
 Antibiotic stock solution can be prepared by commercially
available antimicrobial powders (with given potency). The
amount needed and the diluents in which it can be dissolved
can be calculated by using either of the following formulas to
determine the amount of antimicrobial powder (1) or diluent (2)
needed for a standard solution:

23.  Prepare antimicrobial agent stock solutions at
concentrations of at least 1000 μg/mL or 10
times the highest concentration to be tested,
whichever is greater.

24. Inoculum prepration
Using a loop or swab, transfer colonies as follows.
1. Direct colony suspension method
Pick several colonies from a fresh (18- to 24-h) nonselective agar
plate to broth or 0.9% NaCl.
2. Log-phase method
a. Pick four or five isolated colonies to 3.0 to 5.0 ml of broth.
b. Incubate at 35ºC for 2 to 8 h until growth reaches the turbidity at
or above that of a 0.5 McFarland standard.
For either the log phase or direct colony suspension method,
vortex well and adjust turbidity visually with sterile broth or 0.9%
NaCl to match a 0.5 McFarland standard. Alternatively, standardize
suspension with a photometric device.

25. Antimicrobial concentration for testing
ESBL
Testing more than one antimicrobial agent improves the sensitivity
of ESBLdetection.
For K. pneumoniae, K.
oxytoca, E. coli
Cefpodoxime 4 µg/mL
Ceftazidime 1 µg/mL
Aztreonam 1 µg/mL
Cefotaxime 1 µg/mL
Ceftriaxone 1 µg/mL
For P. mirabilis:
Cefpodoxime 1 µg/mL
Ceftazidime 1 µg/mL
Cefotaxime 1 µg/mL

26. Procedure
 Inoculation: Within 15 minutes after the
inoculum has been standardized as described
above, add 0.1mL of the adjusted inoculum to
each well containing 0.1 mL of antimicrobial
agent in the dilution series (and a positive
control well containing only broth), and mix.
 This results in a 1:2 dilution of each
antimicrobial concentration and a 1:2 dilution of
the inoculums.

27. Interpretation
 After incubation period compare the amount of
growth in the wells containing the antimicrobial
agent with the growth in the growth-control
wells (no antimicrobial agent) used in each set
of tests when determining the growth endpoints.
For a test to be considered valid, acceptable
growth (≥ 2 mm button or definite turbidity) must
occur in the growth-control well.

28. Result
Growth at or above the screening
 For Escherichia coli, Klebsiella
pneumoniae, and Klebsiella
oxytoca
Cefpodoxime ≥ 8 µg/mL
Ceftazidime ≥ 2µg/mL
Aztreonam ≥ 2µg/mL
Cefotaxime ≥ 2µg/mL
Ceftriaxone ≥ 2µg/mL
 For P. mirabilis
Cefpodoxime ≥ 2µg/mL
Ceftazidime ≥ 2µg/mL
Cefotaxime ≥ 2µg/mL

29. QC recommendations
E. coli ATCC 25922 = No Growth
Cefpodoxime 0.25–1 µg/mL
Ceftazidime 0.06–0.5 µg/mL
Aztreonam 0.06–0.25 µg/mL
Cefotaxime 0.03–0.12 µg/mL
Ceftriaxone 0.03–0.12 µg/mL
K. pneumoniae ATCC 700603
= Growth
Cefpodoxime ≥8 µg/mL
Ceftazidime ≥ 2µg/mL
Aztreonam ≥ 2µg/mL
Cefotaxime ≥ 2µg/mL
Ceftriaxone ≥ 2µg/mL

30. Double disc synergy test (DDST)
Mueller-Hinton agar plates were inoculated
with standardized inoculum of the test
organism (corresponding to 0.5 McFarland
tube) with sterile cotton swab.
 A disc of augmentin (20 μg of amoxicillin
and 10 μg of clavulanic acid) is placed in the
middle of the inoculated plate.
 Then 3rd generation cephalosporin discs of
cefotaxime (30 μg), ceftriaxone (30 μg) and
ceftazidime (30 μg) are placed 20 mm
distance from augmentin disc.

31. Double disc synergy test (DDST)
CTX=cefotaxime
AMC= amoxiclav
CAZ=ciftazidime

32. Combined Disk Test / phenotypic
confirmatory disc diffusion test
 In this test, a disk containing cephalosporin
alone(cefotaxime 30μg, or ceftazidime 30μg) is
placed in opposite direction to a disk containing
cephalosporin plus clavulanic acid (30/10μg)
with a distance of 15 mm apart on Muller Hinton
agar medium.
 The inoculated media are then incubated at
37ºC for 18–24 hours. After incubation, zones of
growth inhibition are measured.

33. Result
 A increase in a zone of inhibition of ≥ 5 mm for
a disk containing cephalosporin plus clavulanic
acid compared to a disk containing
cephalosporin alone is considered positive for
ESBL.

34. Phenotypic confirmatory disk diffusion
Test

35. Broth microdilution
 Testing necessitates using both cefotaxime and
ceftazidime,alone and in combination with
clavulanate.
 Antimicrobial concentration:
Ceftazidime 0.25–128 µg/mL
Ceftazidime-clavulanate 0.25/4–128/4 µg/mL
Cefotaxime 0.25–64 µg/mL
Cefotaxime-clavulanate 0.25/4–64/4 µg/mL

36.  Following incubation at 37ºC for 16 hours,
phenotypic confirmation is considered as a ≥ 3
twofold serial dilution decrease in Minimum
Inhibitory Concentration (MIC) of either
cephalosporin in the presence of clavulanic acid
compared to its MIC when tested alone.

37. E-test (Epsilon test)
 The principle behind the ESBL E-test strip is
dilution and diffusion.
 The ESBL E-test strip is a thin plastic, non-
porous strip with dimensions 60mm by 5mm,
and bearing two shorter gradients aligned in
opposite directions on the same strip.
 The strip is two sided containing a gradient of
one of the Oxyiminocephalosporins
(e.g.Ceftazidime alone) on one end and a
gradient of Cephalosporin +Clavulanic
acid(e.g.Ceftazidime plus clavulanate) on the
other.

38. Etest ESBL strip

39. Etest ESBL strip

40. Automated Detection of ESBL
 The VITEK 2 ESBL test (bioMérieux) is a new
tool for rapid detection of ESBL production
which is based on simultaneous assessment of
the inhibitory effects of cefepime, cefotaxime,
and ceftazidime, alone and in the presence of
Clavulanic Acid.
 BD Phoenix:

41.  Microscan ESBL
These are an automated microbial identification
and antibiotic susceptibility testing methods that
can be used to confirm ESBLs in members of
the family Enterobacteriaceae.
 Decrease in the calculated MIC in the presence
of beta lactamase inhibitor is positive for ESBL.

42. Multiplex PCR
 To detect TEM, SHV, OXA, CTX-M, and GES
enzymes in gram-negative bacteria both
conventional PCR as well as real-time PCR
assays have been developed using primers for
one or more ESBL enzymes, and several novel
multiplex formats have recently been used
including microarrays to simultaneously detect a
large panel of β-lactamases.
 A multiplex PCR method has been developed to
classify extended spectrum β-lactamase
(ESBL) and plasmid- mediated AmpC β-
lactamase (PABL).

43. Detection of MBL
1)Imipenem +EDTA combined disk diffusion
method
2)Imipenem-EDTA Double disk Synergy test
(DDST)
3)MBL E-test (Biomerieux)

44. imipenem +EDTA combined disk
diffusion method
 Lawn culture of the test organism is done on
Mueller Hinton Agar plates and Imipenem 10µg
discs are placed on the surface of the agar
plate and 0.5 M EDTA solution is added to one
of the Imipenem disc to obtain a desired
concentration of 750µg.
 Then the inhibition zones of Imipenem and
Imipenem+EDTA discs are compared after 16-
18 h of incubation at 37ºC.
 Zone difference of ≥7mm between Imipenem
alone and with Imipenem+ EDTA interpreted as
positive MBL producing strain.

45. imipenem +EDTA combined disk
diffusion method

46. Imipenem-EDTA Double disk Synergy
test (DDST)
 Test organisms were inoculated on Mueller
Hinton agar plates as recommended by CLSI.
 A 10 µg imipenem discs is placed on the
surface of the agar plate.
 One blank filter paper disk containing 10 μL
EDTA is placed at a distance of 20 mm from the
centre of imipenem disk.
 The inhibition zones of the imipenem and blank
disk with EDTA were compared after 16-18 hrs
of incubation at 35ºC.
 Enhancement of the zone of inhibition in the

47. Imipenem-EDTA Double disk Synergy
test (DDST)

48. MBL E-test
 A 0.5 Mc Farland standard well emulsified
solution of the test strain is plated on a Mueller
Hinton Agar plate by swabbing it by a sterile
swab rotating thrice 60 degrees each to get an
uniform distribution of inoculum.
 A MBL E-test strip is then put carefully on the
agar surface and the plates were incubated at
35ºC in a incubator for 16-18 hrs.
 The result is interpreted as MIC break points by
the zone of intersection around the strip.
 A reduction of IP MIC ≥ 3 twofold dilutions in the

49. MBL E-test

50. Tests Used for Epidemiological or Infection Prevention–
Related Testing

51. mCIM & eCIM test
 mCIM=Modified Carbapenem Inactivation Methods
 eCIM=EDTA Carbapenem Inactivation Methods
 These tests are done for epidemiological or infection
prevention purposes.
 mCIM with or without eCIM testing is not currently
recommended for routine use.
 mCIM is used for detecting carbapenemases in
Enterobacterales and P. aeruginosa whereas eCIM is
used together with mCIM to differentiate metallo-β-
lactamases from serine carbapenemases in
Enterobacterales.
 eCIM is valid only if mCIM is positive.

52. mCIM Test
Test
procedure:
1. For each
isolate to be

53. 6. Inoculate an
MHA plate with
E. coli ATCC ®
25922 as for the
routine disk

54. 8. Invert and incubate the MHA plates at 35°C ±
2°C in ambient air for 18–24 hours.
9. Following incubation, measure the zones of
inhibition as for the routine disk diffusion
method.

55. eCIM for Enterobacterales
 Test procedure:
1. For each isolate, label a second 2-mL TSB
tube for the eCIM test.
2. Add 20 µL of the 0.5 M EDTA to the 2-mL TSB
tube.
3. Follow steps 1 through 9 above as for mCIM
procedure. Process the mCIM and eCIM tubes
in parallel.
4. Place the meropenem disks from the mCIM
and eCIM tubes on the same MHA plate
inoculated with the meropenem susceptible E.

56. Test interpretation [mCIM]
Carbapenemase positive:
 Zone diameter of 6–15 mm or presence of
pinpoint colonies within a 16–18 mm zone.
 If the test isolate produces a carbapenemase,
the meropenem in the disk will be hydrolyzed
and there will be no inhibition or limited growth
inhibition of the meropenem-susceptible E. coli
ATCC 25922.
 Carbapenemase negative:
 Zone diameter of ≥ 19 mm (clear zone)

57. Reference-clsi guidelines M100
 mCIM Results for QC Strains: Negative Control K.
pneumoniae ATCC BAA-1706™ (A) and Positive Control
K. pneumoniae ATCC BAA-1705™ (B).
NOTE: A narrow ring of growth around the meropenem disk
as seen with the negative control (A) results from carryover
of the test organism in the TSB and should be ignored.
B
A

58.  Result: positive mCIM [Pin point colony within
the zone]
 Report: carbapenemase detected
 NOTE: A narrow ring of growth around the
meropenem disk results from carryover of the

59. mCIM and eCIM Test Interpretation: Negative
mCIM. “A” shows an mCIM negative result
(zone diameter = 20 mm) and “B” shows an
eCIM invalid result. Do not interpret the eCIM
result when the mCIM is negative as the isolate
is negative for carbapenemase production.
 Result: negative for carbapenemase production
A B

60.  “A” shows an mCIM positive result (zone
diameter of 6 mm) and “B” shows an eCIM
positive result (zone diameter = 15 mm with
pinpoint colonies throughout the zone of
inhibition).
 NOTE: The pinpoint colonies throughout the
zone of inhibition are ignored when measuring
the zone for the eCIM test. A ≥ 5-mm increase
in zone diameter for eCIM vs zone diameter for
[A] mCIM and [B] eCIM Test
A (6mm) B (15 mm)

61.  “A” shows an mCIM positive result (zone
diameter = 6 mm) and “B” shows an eCIM
positive result (zone diameter = 19 mm). A ≥ 5-
mm increase in zone diameter for eCIM vs
diameter for mCIM zone (19 mm − 6 mm = 13
mm) demonstrates the inhibition of the metallo-
β-lactamase in the presence of EDTA.
 Result: positive mCIM and eCIM
A(6mm) B (19mm)

62.  “A” shows an mCIM positive result (zone
diameter = 6 mm) and “B” shows an eCIM
negative result (zone diameter = 6 mm). Serine
carbapenemases are not inhibited by EDTA and
demonstrate a ≤ 4-mm increase in zone
diameter for eCIM vs zone diameter for mCIM.
 Result: positive mCIM and negative eCIM
 Report: serine carbapenemase detected
A (6mm)
90
B (6mm)
A (mCIM) & B(eCIM)

63.  PCR assays were performed to amplify the
sequences of the-
 BlaIMP
 BlaVIM
 BlaGIM
 BlaSPM
 BlaSIM genes
MBL gene PCR amplification and
sequencing

64. References
 CLSI guidelines M100
 Clinical Microbiology Procedures Handbook 3
edition
 Koneman’s color atlas and textbook of
diagnostic microbiology
 Bailey and Scott's diagnostic microbiology 13th
edition
 Mackie & McCartney Practical Medical
Microbiology 14th edition