To phenotypically screen for the production of metallo-beta-lactamase (MBL) enzyme in the test isolates, all the test organisms should be initially subjected to the three carbapenems which are: imipenem (IPM), meropenem (MEM), and ertapenem (ETP) in order to detect the level or pattern of their susceptibility to these antibiotics as per the Clinical Laboratory Standard Institute (CLSI) criteria.
The screening of the bacterial isolates for the production of MBL enzymes should be conducted by the Kirby-Bauer disk diffusion technique. Each of the single antibiotic disks should be placed at a distance of 25 mm apart, on a Mueller-Hinton (MH) agar plate.
The MH agar plates should be incubated at 37oC for 18-24 hrs. MBL enzyme-producing isolates should be suspected when the test organism(s) is resistant to any of the carbapenems used in the screening test. As per the CLSI criteria, isolates showing inhibition zone diameter (IZD) of ≤ 23 mm for any of the tested carbapenems should be considered and suspected to produce MBL enzyme; and these isolates should be tested further using a phenotypic confirmation test.
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):
- Activity against carbapenem antibiotics.
- No clear hydrolysis of monobactams.
- Inhibition by chelating agents such as ethylene diamine tetraacetic acid (EDTA) and dipicolinic acid.
- 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
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.