Pharmaceutical Microbiology

Test for inducibility of chromosomal β-lactamase (AmpC enzymes)

Written by MicroDok

AmpC enzymes are chromosomally mediated enzymes, and they are produced in bacterial pathogens under induction by some cephalosporins (e.g., ceftazidime and cefotaxime) which induce their production. These chromosomally-mediated enzymes (i.e., AmpC β-lactamases) ultimately destroy, or render ineffective, the antimicrobial onslaught of the antibiotics that stimulate their production by the pathogen.

PROCEDURE FOR PHENOTYPIC DETECTION OF AmpC ENZYMES

AmpC enzyme production should be detected on bacterial isolates resistant to any of the third-generation cephalosporins (ceftazidime and cefotaxime) including the second-generation cephalosporins or cephamycins. Single antibiotic disk containing 30 µg of cefoxitin and another cefoxitin (30 µg) disk impregnated with 5 µl of EDTA (a chelating agent) should be aseptically placed on Mueller-Hinton (MH) agar plate(s) already swabbed with the test bacteria.

Susceptibility or antibiotic disks should be placed at a distance of about 25 mm on the MH agar plate. The MH agar plates should be incubated at 37oC for 18-24 hrs. A difference of ≥ 5 mm inhibition zone diameter around the cefoxitin disk alone and cefoxitin disk+EDTA confirms AmpC enzyme production phenotypically.

AmpC enzyme-producing bacteria are resistant to cefoxitin, amoxicillin-clavulanic acid and third-generation cephalosporins but susceptible to cefepime, a fourth generation cephalosporin. They are also susceptible to imipenem, a carbapenem. Therefore, AmpC enzyme production can also be phenotypically confirmed by evaluating the susceptibility of test pathogens to cefepime.

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.

 

REFERENCE

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.

 

 

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MicroDok

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