INHIBITION BASED ASSAY FOR DETECTION OF METALLO-ΒETA-LACTAMASE

Metallo-β-lactamase and other carbapenemases are inhibited by ethylenediamine tetraacetic acid (EDTA), boronic acid and dipicolinic acid. These agents are all known as chelating agents. Thus, the production of these enzymes by bacteria is detected phenotypically in the laboratory by subjecting the test pathogens to an inhibition based assay that employs any of the above mentioned chelating agents (e.g. EDTA).

Test isolates found to be resistant to imipenem or meropenem (as described by the CLSI breakpoints for MBL screening) should be evaluated phenotypically for metallo beta lactamase (MBL) production. Only after such a phenotypic confirmation test can you infer that the organism tested is MBL positive or negative.

To carry out the inhibition based assay test for the phenotypic detection of MBL, test bacteria (adjusted to 0.5 McFarland turbidity standards) should be aseptically swabbed on Mueller-Hinton (MH) agar plates using a sterile cotton swab.

Imipenem (10 µg), meropenem (10 µg) and ertapenem (10 µg) disks impregnated with EDTA (1 µl) should be aseptically placed on the MH agar plates. And supplementary imipenem (10 µg), meropenem (10 µg) and ertapenem (10 µg) disks without EDTA should also be placed alongside the carbapenem disks encumbered with the chelating agent (EDTA).

The chelating agent (EDTA) should be tested on the test bacteria prior to the inhibition based assay to ensure they have no inhibitory effect on the test organisms. All plates should be incubated at 37oC for 18-24 hrs, and the zones of inhibition recorded using a meter rule. A difference of ≥ 7 mm between the zones of inhibition of any of the carbapenem disks with and without the chelating agents infers metallo-β-lactamase production phenotypically.

BRIEF NOTE ON MBL

Metallo-beta-lactamases (MBLs) are carbapenem-hydrolyzing beta-lactamases which belong to molecular Class B of Ambler beta-lactamase classification, and which have the ability to hydrolyze and confer resistance to carbapenems (imipenem, meropenem, ertapenem) and other beta-lactam antibiotics. Class B carbapenemases (i.e. the metallo-β-lactamases) are found in Enterobacteriaceae, Acinetobacter species and Pseudomonas aeruginosa isolates. MBLs, which are a type of carbapenemases, are an emerging public health problem among clinically important Gram negative organisms including P. aeruginosa, A. baumannii and the Enterobacteriaceae. The carbapenems are very potent antimicrobial agents used for the treatment of serious Gram negative bacterial infections including those that are ESBL-mediated; and because of the broad spectrum activity and stability of the carbapenems to most beta-lactamase enzymes, the carbapenems have been widely used under restricted conditions in most hospitals worldwide as the first-line treatment for severe Gram negative infections. The MBLs are known to confer variable range of high resistance to all beta-lactam antibiotics except the monobactams and their presence in clinically important Gram negative bacteria have put the use of the carbapenems under threat.

The MBLs belong to a group of beta-lactamases which requires divalent cations (e.g. zinc ions) as cofactors for their enzyme activity, and they share four main characteristics as follows (Toleman et al., 2005 and Varaiya et al., 2008):

  1. Activity against carbapenem antibiotics.
  2. No clear hydrolysis of monobactams.
  3. Inhibition by chelating agents such as ethylene diamine tetraacetic acid (EDTA) and dipicolinic acid.
  4. Requirement of zinc ions (Zn2+) for enzyme activity.

Genetically, the MBLs are either plasmid-mediated or chromosomally-mediated, and those that are plasmid-mediated (or encoded by transferable genes or elements such as integrons and transposons) are found in more resistant bacteria such as P. aeruginosa, A. baumannii, and the Enterobacteriaceae while those that are chromosomally-mediated are found in bacterial strains such as Bacillus cereus and Stenotrophomonas maltophilia and in obscure non-clinical bacteria such as Aeromonas species. MBL genes are important resistance determinants considering the fact that most of these genes are carried as mobile gene cassettes (which can easily be integrated into the chromosomes of other susceptible organisms) on class one integrons with the potential to spread to other clinically important bacteria. And because the MBL genes are mainly plasmid-borne, their spread to the population of pathogenic organisms is of great concern and a menace to our ability to fight and treat a wide variety of Gram negative infections

REFERENCE

Chika Ejikeugwu, Charles Esimone, Ifeanyichukwu Iroha, Peter Eze, Malachy Ugwu, Michael Adikwu (2018). Genotypic and Phenotypic characterization of MBL genes in Pseudomonas aeruginosa isolates from the non-hospital environment. Journal of Pure and Applied Microbiology (JPAM), 12(4):1877-1885.  

 

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