DISK ANTAGONISM TEST

INTRODUCTION
• β - lactamases are enzymes produced by bacteria that cause
hydrolysis of the functional β - lactam ring of the β - lactam agents,
rendering them ineffective.
• There are various classification of β – lactamases; Ambler’s
classification of β – lactamases is one of the most common and
accepted one.
Serine β - lactamases Class A Extended Spectrum β - lactamases
Class C Amp C β - lactamases
Class D OXA β - lactamases
Metallo β - lactamases Class B Metallo β - lactamases
INTRODUCTION
• Amp C β - lactamases are Class C Serine β - lactamases group of enzymes that are
resistant to penicillin, 1st, 2nd, 3rd generation cephalosporins, cephamycin, and
β‑lactam/β‑lactamase inhibitor combinations, but not to 4th generation
cephalosporins and carbapenems.
• It was the first bacterial enzyme discovered to destroy penicillin which was seen
in Escherichia coli.
• Stepwise-enhanced resistance in β - lactamases due to mutations were termed
amp A and amp B.
• A mutation in an amp A strain causing reduced resistance was called amp C.
INTRODUCTION
• In many Enterobacteriaceae, Amp C expression is low but inducible in response to
Beta lactam antibiotic exposure. The induction mechanism is very complex.
• Some Enterobacteriaceae, such as Enterobacter species, Citrobacter spp.,
and Serratia spp., carry an inducible AmpC gene.
• In these cases, the gene is strongly induced by β-lactams, such as cefoxitin
and imipenem, with expression mediated by the regulator AmpR.
PRINCIPLE
• In this test, Cefoxitin, imipenem, and amoxicillin-clavulanate serve as potential
inducers of Amp C β – lactamases
• 3rd generation cephalosporin – ceftazidime, aztreonam, and piperacillin-
tazobactam serve as substrates for Amp C β – lactamases.
REAGENTS
• 30 µg cefoxitin disk
• 30 µg aztreonam disk
• 100/10 µg piperacillin-tazobactam disk
• 75/30 μg cefoperazone-sulbactam disk
• 30 µg ceftazidime disk
• Mueller Hinton agar plate
• 0.5 McFarland turbidity standard

SPECIMEN
• Test organism: 18 to 24 hours subculture

BIOSAFETY LEVEL
•
PROCEDURE
• Prepare 0.5 McFarland bacterial suspension from an overnight blood agar plate.
• Inoculate the surface of the Mueller Hinton agar plate using this suspension as
per the standard disk diffusion method.
• Place a 30-µg cefoxitin disk at the center of the plate.
• Place 30 µg aztreonam disk, 100/10 µg piperacillin-tazobactam disk, 75/30 μg
cefoperazone sulbactam disk and 30 µg ceftazidime maintaining a distance of 20
mm between each disk.
• Invert the plate and incubate overnight at 35°C - 37°C.
PLATE READING AND INTERPRETATION
• After overnight incubation, examine the plate for any obvious blunting or
flattening of the zone of inhibition between the aztreonam disk, piperacillin-
tazobactam disk, cefoperazone- sulbactam disk, ceftazidime and the inducing
substrate (cefoxitin disk)
• If there is any blunting or flattening of the zone, consider as a positive result for
AmpC production
PLATE READING AND INTERPRETATION

Flattening of zones of aztreonam disk, piperacillin-tazobactam disk, cefoperazone-

sulbactam disk, and ceftazidime disk toward cefoxitin disk (inducing substrate)
showing a positive result.
REFERENCES
• M100Ed33 - Clinical and Laboratory Standards Institute (CLSI), Performance
Standards for Antimicrobial Susceptibility Testing - 33rd edition.
• Essentials of antimicrobial stewardship - Dr. Apurba Sastry - 1st edition