EDTA Disks

Used in the antimicrobial susceptibility testing for the phenotypic detection of metallo ß lactamase enzymes (MBLs).

Purpose

Metallo ß lactamase enzymes (MBLs) belong to group 3 according to the functional classification of Bush-Jacoby-Medeiros (Ambler class B) and are inhibited by chelating agents of zinc such as EDTA and sodium mercaptoacetate.

They are chromosomally or plasmid encoded, and have emerged in many geographical locations (including Argentina) often conferring high-level resistance to beta lactams including penicillins, cephalosporins (1st, 2nd, 3rd and 4th generation) and carbapenems except aztreonam.
The clinical utility of carbapenems is under threat with the emergence of MBLs, because these antimicrobial agents are stable against the action of Extended Spectrum Beta Lactamses (ESBLs) and are used for the treatment of diseases caused by gram negative bacteria resistant to beta lactams.
Due to the worldwide increase in the occurrence, rate of dissemination and the ability of MBLs to rapidly disseminate within an institution leading to poor outcomes when infection ensues is a concern, early laboratory detection is critical and of great clinical importance.
So, practical and simple, highly sensitive and specific phenotypic methods for the identification of bacteria producing MBLs are useful in surveillance studies and to control infection diseases.

Composition of EDTA Disks

EDTA (Ethyllenediaminetetraacetic acid): 372 ug.

Sodium mercaptoacetate: 900 ug.

Methodology:

Disk Diffusion Method on Mueller Hinton agar according to CLSI standards and guidelines (*)

For the detection of metallo ß lactamase enzymes (MBLs), perform the Double Disk Synergy Test detailed below:
Place one disk of EDTA on the surface of Mueller Hinton Agar plate on which the bacterial suspension to be examined had been spread and one disk containing 10 ug of imipenem with a edge to edge distance to the EDTA disk of 15 mm.
Then, place one disk containing 10 ug of meropenem with edge to edge distance to the EDTA disk of 15 mm, opposite to the disk of imipenem.

Results

Positive test: the change in the shape of the growth-inhibitory zone around the disk of imipenem (10 ug) or meropenem (10 ug) through the interaction with the EDTA disk is observed for the detection of metallo ß lactamase production.

Negative test: absence of change in the shape of the growth-inhibitory zone around the carbapenem antimicrobial agent through the interaction with the EDTA disk.

Storage

Between -20 and 0 °C until expiration date.
It is possible to store disks at 2-8°C for up one week.
Allow containers of disks to reach 10-35 º C before opening them.

Packaging

50 disks.
Code: B1241727.

Disk Diffusion Method on Mueller Hinton agar according to CLSI standards and guidelines (*)

Preparation of plates containing the test culture medium

Mueller Hinton agar. Prior to use, check its pH is between 7.2 and 7.4.
Pour no more than 25 to 30 mL of sterile melted culture medium into Petri dishes, ensuring 4 mm. in medium depth.
If just before using, excess of moisture is present on the surface of the culture medium, place plates in an incubator at 35-37º C approximately 30-60 minutes to dry them. 

Test sample:

Pick 3-5 well isolated colonies of the organism to test and transfer them into a tube containing 5 mL of Tryptein Soy Broth. Adjust inoculum density to 0.5 Mc Farland scale.

Preparation of Mc Farland Standars: add 0.5 mL of BaCl2 0.048 M (1.175 P/V BaCl2.2H20) to 99.5 ml H2S04 0.36 N (1 % V/V).

lnoculation of test plates.

Dip a sterile non-toxic swab into the adjusted suspension. Rotate the swab several times pressing firmly on the inside wall of the tube above the fluid level.

Inoculate the dried surface of the agar medium by streaking the swab over the entire agar surface. Repeat this streaking procedure twice, rotating the plate approximately 60 degrees each time. Replace the plate top and allow 3 to 5 minutes for any surface moisture to be absorbed before placing disks mentioned above.

Disk application.

Place the appropriated disks using sterile forceps. Gently press down each disk to ensure complete contact with the agar surface.
Allow to stand the plates, and after 15 minutes incubate them in inverted position at 35-37 ºC overnight (during 18-24 hours).

References