Pharmaceutical Microbiology

AmpC ENZYMES

Written by MicroDok

AmpC beta-lactamase enzymes began to gain importance in the health sector in the early 1970s as one of the mediators of antimicrobial resistance in Gram-negative bacteria including Escherichia coli and Pseudomonas aeruginosa. 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. Inducible AmpC beta-lactamases are usually seen in Citrobacter species, Morganella species, Enterobacter species and Serratia marcescens while the plasmid-mediated AmpC enzymes are seen in other enteric and non-enteric bacteria including Escherichia coli, Klebsiella species and Pseudomonas aeruginosa.

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 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 stable 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.

Thus, the genes for AmpC enzyme production can be transferred to other susceptible bacteria from organisms harbouring them through plasmids. Klebsiella pneumoniae is one of the few Gram-negative bacteria which do not possess a chromosomal AmpC beta-lactamase gene, but the organism can still acquire the gene for the enzyme production via the transfer of AmpC-containing plasmids from another AmpC-beta-lactamase positive bacteria. AmpC enzymes are intrinsic cephalosporinases found on the chromosomal DNA of many Gram-negative bacteria and opportunistic bacteria; and they confer resistance to a wide array of antibiotics including penicillins, 2nd and 3rd generation cephalosporins, cefoxitin and beta-lactamase inhibitors such as amoxicillin-clavulanic acid. However, AmpC enzyme-producing bacteria are still susceptible to the carbapenems and fourth generation cephalosporins (e.g. cefepime).

According to the European Food Safety Authority (EFSA), the concern today around the world that is of public health importance is not that AmpC positive bacteria exist but that a growing number of the AmpC enzymes have escaped on to plasmids and can be transferred to other bacteria within the environment or hospital setting. The failure to detect AmpC beta-lactamase enzymes has no doubt contributed to the uncontrolled spread of AmpC positive bacteria; and in most of the cases these has also contributed to the treatment failures experienced in patients infected with such organisms. Of particular concern are the limited treatment options for infections caused by Gram-negative resistant bacteria – leading to antibiotic selection pressure and consequent risk of the emergence of antibiotic resistant pathogens. Studies have shown that the onslaught of AmpC resistance represents a major challenge for physicians as these high-profile beta-lactamase hydrolyzing enzymes renders third-generation cephalosporins and the cephamycins increasingly inefficacious in the treatment of bacterial related infections caused by AmpC-producing bacteria.

The detection of AmpC beta-lactamases in bacterial isolates still remains problematic especially in those organisms that produce or have extended spectrum beta-lactamases (ESBLs); and this is due in part to the fact that ESBL-producing bacteria that also harbour genes for AmpC enzyme production mask the production of AmpC enzymes. This is why the Clinical Laboratory Standard Institute (CLSI) and other researchers recommend the use of several antimicrobial agents that also incorporates chelating agents such as ethylene diamine tetraacetic acid (EDTA) and boronic acid for the detection of AmpC enzymes from both environmental and hospital isolates. It is even more worrisome when clinical microbiology laboratories in some hospitals fail to detect these pathogens from clinically important specimens and/or other environmental samples.

Bacterial resistance to the cephalosporins (especially 3rd-generation cephalosporins) and the cephamycins should raise a suspicion for possible production of AmpC beta-lactamases – that warrants phenotypic confirmation. The confirmation of AmpC production in clinical and/or environmental pathogens is important for the patient’s welfare because it will support the susceptibility test result by permitting the reservation of broad spectrum antibiotics (e.g. carbapenems) for more serious and complicated bacterial disease. And such a confirmatory test coupled with the antimicrobial susceptibility test results will help to select targeted narrow spectrum antibiotics for treatment rather than using drugs with broad spectrum activity, thereby minimizing the risk of selecting for, or promoting the development and spread of antimicrobial resistant bacteria pathogens in either the community or hospital environment.

Chromosomal AmpC enzymes

Chromosomal AmpC enzymes are AmpC beta-lactamases that are mediated by AmpC genes – which is usually located on the chromosomal DNA of the host bacteria. The ampC genes are intrinsically located in the DNA of bacteria producing these enzymes; and they mediate the hyper-production of AmpC beta-lactamases in pathogenic bacteria. As aforementioned, bacterial organisms that harbour the AmpC genes include Enterobacter species, Serratia marcescens, Citrobacter freundii, Morganella morganii, Hafnia species and Providencia species. Escherichia coli and Shigella sonnei contain a chromosomal AmpC gene but due to a lack of the regulatory gene AmpR, the AmpC gene in E. coli and S. sonnei is not fully expressed in amounts large enough to confer resistance as is applicable in the case of the other organisms that hyper-produce AmpC beta-lactamases (C. freundii, S. marcescens and Enterobacter species).

Though AmpC beta-lactamases are produced at low levels and may not cause resistance, these enzymes can still be produced at very high levels in other bacteria and cause resistance. Chromosomal AmpC beta-lactamases can be produced inducibly or constitutively. The inducible expression of AmpC gene occurs when the AmpC beta-lactamase is produced at a high level; and this inducibility usually occurs when the bacterial organism is exposed to inducing agents such as cephamycins and other broad-spectrum beta-lactam agents that drive its production. However, this induction is usually temporary and the production of AmpC enzymes in such scenarios may be reversed when the antimicrobial agent inducing its production in the pathogens environment is removed.

However, when mutation occurs in bacterial organisms producing AmpC beta-lactamases, the AmpC gene becomes permanently expressed at very high levels; and such pathogens are called permanently depressed mutants. These permanently depressed mutants of AmpC producers harbour plasmids that encode the genes responsible for the enzyme production; and these resistant traits can be passed on to susceptible bacteria in the environment through genetic transfer mechanisms such as conjugation or transformation.

Plasmid-mediated AmpC enzymes

Plasmid-mediated AmpC beta-lactamases are beta-lactamases produced by bacterial pathogens devoid of chromosomal AmpC genes but that may have acquired resistance plasmids of the resistance traits via antibiotic selection pressure, mutation or through gene transfer mechanisms. Plasmid-mediated AmpC beta-lactamases and/or genes have been known since 1989; and they have also been discovered around the world from both clinical and non-clinical bacterial isolates. The plasmid-mediated AmpC beta-lactamases unlike the chromosomal AmpC enzymes confer resistance to a broad spectrum of beta-lactams including ampicillin, piperacillin, temocillin, cephalothin, cefotaxime, ceftazidime, cefoxitin, cefotetan, cefmetazole, moxalactam, aztreonam, cefepime, cefpirome, imipenem, and meropenem.

They are usually found in bacterial organisms that do not naturally carry the chromosomal AmpC gene. Plasmid-mediated AmpC beta-lactamases have also been detected in Klebsiella species, Escherichia coli, Proteus species and Salmonella species. Plasmid-mediated AmpC beta-lactamases were first recognized in the early 1980s, and since then, these phenotypic traits of antimicrobial resistance have spread among members of the family Enterobacteriaceae and even to non-enteric bacteria such as P. aeruginosa. Epidemiological studies have shown that AmpC enzyme producing bacteria are recovered from hospitalized patients after several days of admission to the hospital; and affected patients have often had prolonged stays in intensive care units (ICUs) and other high dependency units of hospitals.

Genes encoding the production of AmpC beta-lactamases in bacteria are found on transferable plasmids, and these genetic elements can be transferred to other sensitive bacterial isolates in an environment. Thus, the determination of AmpC enzyme production from both clinical and environmental isolates is important because such a practice will help to reduce the incidence of the emergence of more resistant strains of bacteria, reduce the spread of resistant pathogens in the community and/or environmental settings, and thus help to guide physicians on the best choice of antimicrobial therapy to manage bacterial infections or diseases caused by these organisms and other resistant bacterial infections.

REFERENCES

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Coudron P.E (2005). Inhibitor – based methods for detection of plasmid – mediated Amp C β – lactamases in Klebsiella spp., Escherichia coli and Proteus mirabilis. J Clin Microbiol, 43:4163-7.

Jacoby G.A (2009). AmpC β-lactamases. Clinical Microbiology Review, 22(1):161-182.

Jacoby G.A and Munoz-Price L.S (2005). Mechanisms of Disease: The New β – Lactamases. N Engl J Med, 352:380-91.

Perez-Perez F.J and Hanson N.D (2002). Detection of plasmid-mediated AmpCβ-lactamase         genes in clinical isolates by using multiplex PCR. Journal of Clinical Microbiology, 40(6):2153-2162.

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