Detection of AmpC β Lactamases in Gram-negative Bacteria

Abstract

AmpC β-lactamases are clinically important cephalosporinases encoded on the chromosomes of many Enterobacteriaceae and a few other organisms, where they mediate resistance to cephalothin, cefazolin, cefoxitin, most penicillins, and β-lactamase inhibitor/β-lactam combinations. The increase in antibiotic resistance among Gram-negative bacteria is a notable example of how bacteria can procure, maintain and express new genetic information that can confer resistance to one or several antibiotics. Detection of organisms producing these enzymes can be difficult, because their presence does not always produce a resistant phenotype on conventional disc diffusion or automated susceptibility testing methods. These enzymes are often associated with potentially fatal laboratory reports of false susceptibility to β-lactams phenotypically. With the world-wide increase in the occurrence, types and rate of dissemination of these enzymes, their early detection is critical. AmpC β-lactamases show tremendous variation in geographic distribution. Thus, their accurate detection and characterization are important from epidemiological, clinical, laboratory, and infection control point of view. This document describes the methods for detection for AmpC β-lactamases, which can be adopted by routine diagnostic laboratories.

Keywords: AmpC β-lactamases; disk approximation test; gram-negative bacteria; three-dimensional extract test.

Figure 1

Representation of three dimensional extract test. (a) Zone of inhibition showing positive test, (b) no zone of inhibition showing negative test. *30 μg cefoxitin disk

Figure 2

Representation of AmpC disk test. (a) Zone of inhibition showing positive test results, (b) no zone of inhibition showing negative test results, *AmpC disks (filter paper containing tris-EDTA), CX: 30μg cefoxifin disks

Figure 3

Representation of boronic acid disk test. A: 30 μg cefoxitin disk supplemented with 300μg of phenyl boronic acid. B: 30 μg cofoxitin disk alone

Figure 4

Representation of disk approximation test. Flattening of zone of ceftazidime toward imipenem disk (inducing substrate) showing positive result. IMP: Imipenem (10 μg), CAZ: Ceftazidime (10 μg), AMC: Amoxillin-clavulanate (20/10 μg)