Detection of AmpC β Lactamases producing Enterobacteriaceae

Overview

AmpC β Lactamases is one of the clinically important causes of drug-resistant in Enterobacteriaceae. the first isolates producing acquired AmpC were identified at end of 1980, and since then they have been observed globally.  Detection of organisms producing this enzyme can be difficult, there is no standard guideline available at present. these enzyme detection and characterization are important in epidemiological and clinical. Three different phenotypic tests to detect AmpC β Lactamases producing organisms.

Definition

AmpC-type cephalosporins are ambler class C β -Lactamases. they hydrolyze penicillins, monobactams, and cephalosporins including the third-generation but not the fourth-generation compounds. In general, AmpC- type enzyme are poorly inhibited by classical ESBL inhibitors, especially clavulanic acid.

Clinical importance of AmpC detection

The major producer species of acquired AmpCs are E.coli, K. pneumoniae, K. oxytoca, Salmonella enterica, and P. mirabilis. Isolates with this enzyme have been recovered from both hospitalized and community patients, and they were recognized earlier than classical ESBL- enzymes in farm animals and in food products (E.coli and Salmonella enterica). although the acquired AmpCs have been spread widely and horizontal transfer of AmpC genes (often referred to as plasmid-mediated AmpC). The AmpC genes originating from natural producers, namely the Enterobacter group (MIR, ACT), the Citrobacter freundii group (CMY-2-like, LAT, CFE), the Morganella morganii group (DHA), the Hafnia alvei group (ACC), the Aeromonas group (CMY-1- like, FOX, MOX) and the Acinetobacter baumanni group (ABA). The most prevalent and most widely disseminated are the CMY-2-like anzymes, although the induible DHA-like β- lactamases and some others have also spread extensively.

Mechanisms of Resistance

Numerous Enterobacteriaceae and other some Gram-Negative bacilli produce natural AmpCs, either constitutively at a trace level or inducibly. The derepression or hyperproduction of natural AmpCs are due to various genetic changes and confer high-level resistance to cephalosporins and to penicillin- β- lactamase inhibitor combinations. The class C cephalosporinses can also occur as acquired enzymes, mainly in Enterobacteriaceae. except for a few inducible type (DHA), the acquired AmpCs are expressed constitutively, conferring resistance similar to that in the derepressed or hyperproducing mutants of natural AmpC producer. Resistance levels depend on the amount of enzymes expressed as well as the presence of other resistance mechanisms like ESBL,

Detection of AmpC refers to the identification or measurement of AmpC beta-lactamase enzymes. AmpC beta-lactamases are a type of enzyme produced by some bacteria that can confer resistance to certain antibiotics, particularly beta-lactam antibiotics like penicillins, cephalosporins, and related drugs.

Recommended methods for detection of acquired AmpC in Enterobacteriaceae

Phenotypic Detection: This involves testing the bacteria's ability to hydrolyze (break down) specific beta-lactam antibiotics. One commonly used method is the "Cefoxitin Disk Diffusion Test." In this test, a bacterial culture is spread on an agar plate, and a disk containing cefoxitin, a specific antibiotic, is placed on the plate. If the bacteria produce AmpC enzymes, they will be resistant to cefoxitin, leading to a larger zone of inhibition around the disk.

AmpC Induction Test: AmpC beta-lactamase expression can be induced by certain conditions, such as growth in the presence of beta-lactam antibiotics. By growing the bacteria in the presence of a known inducer (e.g., cloxacillin) and then testing their resistance to various beta-lactam antibiotics, the increased resistance can indicate the presence of AmpC.

PCR (Polymerase Chain Reaction): Molecular techniques like PCR can be used to detect the genes that encode for AmpC beta-lactamases. By isolating the bacterial DNA and amplifying specific gene regions using PCR, the presence of AmpC genes can be confirmed.

Multiplex PCR: This technique allows for the simultaneous detection of multiple genes, including different variants of AmpC beta-lactamases, in a single reaction. This is particularly useful as there are several different types of AmpC enzymes.

Sequencing: DNA sequencing can be used to identify specific mutations in the AmpC genes. This can help determine the type of AmpC enzyme present and whether it might confer resistance to specific antibiotics.

Phenotypic Confirmatory Tests: Since AmpC enzymes can sometimes be challenging to differentiate from other types of beta-lactamases, additional tests may be required to confirm the presence of AmpC. These tests might involve the use of inhibitors that selectively target other beta-lactamase enzymes, leaving AmpC activity unmasked.

Appropriate strains for quality control of AmpC detection tests

Strain	Mechanism
E. coli CCUG 58543	Acquired CMY-2 AmpC
E. coli CCUG62975	Acquired CMY AmpC and CTX-M-1 group  ESBL
E. coli ATCC 25922	AmpC negative.

References

Empel J, Hrabák J, Kozioska A, Bergerová T, Urbášková P, Kern-Zdanowicz I, Gniadkowski M. DHA1-producing Klebsiella pneumoniae in a teaching hospital in the Czech Republic. Microb Drug Resist. 2010;16:291-295

 D’Andrea MM, Literacka E, Zioga A, Giani T, Baraniak A, Fiett J, Sadowy E, Tassios PT, Rossolini GM, Gniadkowski M, Miriagou V. Evolution of a multi-drug-resistant Proteus mirabilis clone with chromosomal AmpC-type cephalosporinases spreading in Europe. Antimicrob Agents Chemother. 2011;55:2735-2742

Bauernfeind A, Schneider I, Jungwirth R, Sahly H, Ullmann U. A novel type of AmpC β-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia. Antimicrob Agents Chemother. 1999;43:1924-31.