Cefoxitin-cloxacillin double-disk synergy test (CC-DDST) is another method that can be used to phenotypically confirm the production of AmpC enzymes in test bacterial isolates that showed reduced susceptibility or resistance to any of the tested antibiotics used in the screening test for AmpC enzyme production. The antibiotics used for screening bacterial isolates for AmpC enzyme production include third-generation cephalosporins (e.g., ceftazidime) and cephamycins (e.g., cefoxitin). Bacteria that produce AmpC-enzymes are usually resistant to these classes of antibiotics.
PROCEDURE FOR PHENOTYPIC DETECTION OF AmpC ENZYMES
CC-DDST test is a phenotypic test for AmpC enzyme detection; and the test is based on the inhibitory effect of cloxacillin on AmpC enzyme. Single disks containing 30 µg of cefoxitin should be placed at a distance of 20 mm away from a disk containing 200 µg of cloxacillin on Mueller-Hinton (MH) agar plates already inoculated with the test bacteria (equivalent to 0.5 McFarland turbidity standards).
The MH agar plates should be incubated at 37oC for 18-24 hours; and the zones of inhibition should be recorded as per the Clinical Laboratory Standard Institute (CLSI) criteria. A difference of ≥ 4 mm in the cefoxitin-cloxacillin inhibition zones minus the cefoxitin disk used alone is indicative of AmpC enzyme production phenotypically.
Brief note on AmpC enzymes
Chromosomal AmpC enzymes (which can also be called inducible AmpC enzymes) and plasmid-borne AmpC enzymes are the two main types of AmpC beta-lactamases that exist amongst bacteria especially in Gram negative organisms – in which these multidrug resistant enzymes are produced. AmpC enzymes are broad-spectrum beta-lactamase enzymes that are usually encoded on bacterial chromosome, and which are active on cephamycins (e.g. cefoxitin and cefotetan) and oxyimino-β-lactam agents. They can also be plasmid encoded; and AmpC enzymes like other extended or expanded beta-lactamases such as ESBLs and MBLs confer on pathogenic Gram negative bacteria the exceptional ability to be resistant to a wide array of beta-lactam drugs and non-beta-lactams. AmpC beta-lactamases are bacterial enzymes that hydrolyze third-generation extended spectrum cephalosporins (e.g., ceftazidime) and cephamycins (e.g., cefoxitin), thus engendering antimicrobial resistance to these categories of antibiotics.
AmpC beta-lactamases are differentiated from extended spectrum beta-lactamases (ESBLs) by the ability of the former (i.e., AmpC enzymes) to hydrolyze cephamycins (e.g., cefoxitin) and their lack of inhibition by clavulanic acid. The expression of AmpC enzyme is typically inducible in several Enterobacteriaceae and other Gram negative bacteria including but not limited to Escherichia coli, Klebsiella species, Enterobacter species and Pseudomonas aeruginosa; and the production of this enzyme facilitates the emergence under antibiotic pressure of highly resistant but stably depressed mutants of the organisms. And these highly resistant but stably depressed mutants of the organisms have the ability to hydrolyze extended spectrum cephalosporins and other beta-lactam agents even though they may still remain susceptible to the carbapenems (e.g., imipenem and meropenem). The genes that codes for the production of AmpC enzymes in bacteria are normally chromosomally-mediated. Plasmid-mediated AmpC enzyme production in bacteria is also possible amongst bacterial organisms through genetic transfer mechanisms such as conjugation and transduction.
Ejikeugwu Chika, Esimone Charles, Iroha Ifeanyichukwu, Adikwu Michael (2018). First Detection of FOX-1 AmpC β-lactamase gene expression among Escherichia coli isolated from abattoir samples in Abakaliki, Nigeria. Oman Medical Journal, 33(3):243-249.