Boronic Acid 300 ug Disks

Used in the antimicrobial susceptibility testing for the detection of AmpC β-Lactamase enzymes.

Purpose

β-Lactamase production is the main mechanism underlying resistance to β-lactam antibiotics in gram negative bacilli.
AmpC β-lactamase enzymes, which belong to group 1 according to the classification of Bush et al (Ambler class C), are cephalosporinases poorly inhibited by β-lactamase inhibitors such as clavulanic acid and sulbactam, but are inhibited specifically and in a reversible way by boronic acids.
These enzymes are clinically important because they confer resistance to a variety of β-lactams, including aminopenicillis , aminopenicillis / β-Lactamase inhibitors, cephalosporins, oxyiminocephalosporins and some cephamycins.
There are chromosomally encoded and inducible AmpC enzymes in several species of Enterobacteriaceae, including Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Morganella morganii.
In the late 1980s, plasmid mediated AmpC beta lactamases were reported in other Enterobacteriaceae members such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Salmonella spp.

Thus, the resistance to cephamycins (cefoxitin) in bacteria lacking of chromosomic AmpC β-lactamase enzymes may suggest the presence of plasmid mediated Amp C β-lactamase enzymes, it also can be due toa decreasing of permeability in outer membrane of bacteria. In addition, resistance to 3rd generation cephalosporins may be due to hyperproduction of AmpC enzymes or extended spectrum beta lactamases (ESBL) enzymes. Each mechanism of resistance have different epidemiology and therapy.

So, practical and simple, highly sensitive and specific phenotypic methods for the identification of bacteria producing AmpC β-Lactamase enzymes are useful in surveillance studies and to control infection diseases.

Methodology:

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

For the detection of AmpC β-lactamase enzymes, perform the Double Disk Synergy Test detailed below:

Place one disk of boronic acid 300 ug on the surface of Mueller Hinton Agar plate on which the bacterial suspension to be examined had been spread and one disk containing 30 ug of cefoxitin with a centre to centre distance to the boronic acid disk of 20 mm.
Then, place one disk containing 30 ug of cefotaxime with a centre to centre distance to the boronic acid disk of 20 mm, opposite to the disk of cefoxitin.

Results

Positive test: the change in the shape of the growth-inhibitory zone around the β-lactam antimicrobial agent through the interaction with the boronic acid 300 ug disk is observed for the detection of AmpC betalactamase production.

Negative test: absence of change in the shape of the growth-inhibitory zone around the β-lactam antimicrobial agent through the interaction with the boronic acid 300 ug 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: B1241627.

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