ESBL production was confirmed among
the suspected bacterial strain according to the gudelines of CLSI (Clinical and
Laboratory Standard Institute) for phenotypic confirmatory testing. According
to these guidelines, when confirming ESBL production among the suspects using
Combined Disk (CD) assay, an increase in zone size of ≥5 mm from either of the
combination disk i.e. clavulanate containing disk indicates the presence of
ESBL in the test organism.
•
The suspected organism was
inoculated into Mueller hinton broth and incubated at 370C until the
turbidity matched 0.5 Mc Farland atandard.Using a sterile cotton swab the test
orgaism was carpet cultured on a MHA plate.
•
With the help of sterile forcep, the
ESBL detection discs were placed onto the inoculated medium ensuring that they
are evenly spaced.
•
The plate was incubated at 35-370C
for 18-24 hours and the results interpreted.
Interpretation
of result
Compare the zone of inhibition for
the ceftazidime and cefotaxime discs to
that of the ceftazidime and cefotaxime plus clavulanic acid combination discs.
An increase in zone diameter of ≥5 mm in the presence of clavulanic acid from
any or all of the discs indicates the presence of ESBL in the test organism.
Quality control
Check
for sign of deterioration. Quality control mst be performed with at least one
organism to demonstrate positive reaction and at least one to demonstrate
negative reaction.
Positive Control:
Escherichia coli NCTC 13351, Klebsiella pneumoniae ATCC 700603
Negative Control:
Escherichia coli ATCC 25922
2. Confirmation of MBL production
using Imipenem-EDTA combined disk test
Currently
there are no Clinical and Laboratory Standard Institute (CLSI) criteria for the
phenotypic detection of MBL production. MBL detection thus depend on the
ability of the chelating agent like EDTA and thiol based compounds which remove
the zinc from active site of the enzyme
thus inhibiting its activity. In this study, we used a comparatively simple
method for the detection of MBL production based on the bacterial genera to be tested
for MBL production.
•
For
Acinetobacter spp. and Pseudomonas aeruginosa
•
A suspension of the test organism
equivalent in density to a Mc Farland 0.5 opacity standard was prepared using a
pure and fresh culture.
•
Using a sterile swab, test organism
was carpet cultured on Mueller Hinton Agar.
•
With the help of sterile forcep two
Imipenem (30µg) disks were placed at 4-5
cm distance apart.
•
To one of the discs 10µl of 0.5M EDTA solution was added
aseptically.
•
The plate was incubated at 35-370C
for 18-24 hours.
•
•
Inetrpretation
of result
•
The plate is observed for the
increase in inhibition zone of the Imipenem-EDTA disk as compared to Imipenem
Disk alone. The increase in zone size of ≥5mm is positive for MBL production.
•
•
For
Enterobacteriaceae and others:
•
A suspension of the test organism equivalent
in density to a Mc Farland 0.5 opacity standard was prepared using a pure and
fresh culture.
•
Using a sterile swab, test organism
was carpet cultured on Mueller Hinton Agar.
•
With the help of sterile forcep two
Imipenem (10µg) disks were placed at 4-5 cm distance apart.
•
To one Imipenem disk 10µl of 100mM
EDTA solution was added aseptically.
•
The plate was incubated at 35-370C
for 18-24 hours.
•
•
Inetrpretation
of result
•
The plate is observed for the
increase in inhibition zone of the Imipenem-EDTA disk as compared to Imipenem
Disk alone. The increase in zone size of ≥5mm is positive for MBL production.
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