Metallo-beta-lactamases (MBLs) are beta-lactamase enzymes produced by pathogenic bacteria, and which hydrolyzes the carbapenems (e.g. imipenem, meropenem, and ertapenem) and render the antibiotics ineffective for treatment. They are encoded by genes that have been procured by pathogenic bacteria either by mutation or by horizontal gene transfer from other resistant microbes. MBLs efficiently hydrolyze all beta-lactam drugs except aztreonam, a monobactam. However, they confer variable range of high levels of resistance to all beta-lactam antibiotics and some non-beta-lactams such as fluoroquinolones and aminoglycosides; and their presence in clinically important Gram negative bacteria have put the use of the carbapenems under threat. MBL enzymes have high affinity for zinc ions (Zn2+); and thus the enzyme is largely inhibited by chelating agents (e.g. EDTA and dipicolinic acid) in vitro.

The susceptibility of MBL-producing bacteria to chelating agents is the basis upon which the enzyme can be detected phenotypically in pathogenic microorganisms. Genes responsible for the expression of MBLs, multidrug-hydrolyzing enzymes can also be chromosomally or plasmid-mediated as is also applicable with extended spectrum beta-lactamases (ESBLs) and other resistance genes harboured by pathogenic microorganisms. The carbapenems as earlier listed are often the last line of treatment option for a variety of infectious disease including those caused by multidrug resistant organisms (e.g. ESBL positive bacteria). The carbapenems are very potent antimicrobial agents used for the treatment of serious Gram negative bacterial infections, and because of the broad spectrum activity and stability of the carbapenems to most beta-lactamase enzymes, they have been widely used under restricted conditions in most hospitals worldwide as the first-line treatment for severe Gram negative infections.

MBLS are mainly expressed by non-lactose fermenters such as the Pseudomonas species and Acinetobacter species. The Enterobacteriaceae including Escherichia coli and Klebsiella species have also been reported to express MBLs. The presence or occurrence of MBL-producing bacteria in a hospital setting poses not only a therapeutic problem but also serious concern for infection control management in the health system. This is due to the fact that organisms producing MBLs are multidrug resistant in nature; and they limit therapeutic options for treatment. MBLs may be disseminated in hospital environment through genetic transfer elements such as transposons, plasmids and integrons amongst clinically important bacteria; and the extensive use of the carbapenems especially irrationally has given impetus to the spread of organisms that produce the enzymes. Pathogenic bacteria producing metallo-beta-lactamases (MBLs) now occur worldwide, and hospital infections have been reported in Europe, Asia, and America and in Africa.

The detection of MBL-producing bacteria is usually two-folds and involves a phenotypic screening test (Figure 1) and confirmatory tests as well as the application of PCR technique to amplify MBL genes in clinically important bacterial isolates. The phenotypic detection methods for MBLs from clinically important Gram negative bacteria make use of tests such as disk approximation, disk diffusion, micro-dilution test, E-test, and carbapenem hydrolysis test – all of which are usually performed based on routine clinical microbiology antimicrobial susceptibility test with some modifications. Particularly, the susceptibility of the test pathogen to any of the carbapenems (e.g. imipenem) is first evaluated by the Kirby-Bauer disk diffusion test; and organisms found to be resistant to any of these agents (based on the breakpoints of the Clinical Laboratory Standard Institute, CLSI) is subjected to a phenotypic confirmation test for MBL detection.

Figure 1. Screening of a clinical isolateof P. aeruginosa for susceptibilty to imipenem (10 µg) and meropenem (10 µg). The P. aeruginosa is highly resistant (R) to the imipenem and meropenem antibiotics (Oxoid, UK). Reduced susceptibility or resistance (R) to either of the carbapenems (imipenem or meropenem) suggests possible MBL production which should be confirmed by either the Modified Hodges test (MHT) or the inhibitor based assay (disk potentiation test) for phenotypic confirmation of MBL production in a test bacterium.

The disk potentiation or inhibitor-based assays (Figure 2) and the modified Hodges test (Figure 3) and are usually used for the phenotypic confirmation of MBL production in bacteria. In the inhibitor based test, MBL production in the test pathogen is detected phenotypically by using the carbapenem alone and in combination with a chelating agent (e.g. imipenem-EDTA). An increase in the zone diameter of any of the carbapenems when used alone and in combination with a chelating agent (i.e. imipenem-EDTA for example) infers MBL production phenotypically. Proper antimicrobial susceptibility testing (AST) is critical for the containment of MBL-producing bacteria, and AST will also help to guide therapy as well as prevent the emergence and spread of MBL genes amongst clinical and environmental isolates. 

Figure 2. Disk potentiation/Inhibitor based assay for the phenotypic confirmation of MBL production in clinical isolates. The test isolate is a clinical strain of P. aeruginosa, and the illustration shows the inhibition of MBL activity in the test isolate by EDTA. The test is positive for MBL production in P. aeruginosa Isolate (No.P33) phenotypically. The plate shows zone of enhancement around imipenem plus EDTA and meropenem plus EDTA. A 7 mm increase in zone diameter of any of the carbapenems (imipenem 10 µg and meropenem 10µg) when used alone and in combination with each imipenem and meropenem disks containing 5 µl EDTA infers MBL production phenotypically.
KEY: IPM-imipenem 10 µg, MEM-meropenem 10µg, EDTA (ethylenediamine tetraacetic acid).

Figure 3. Modified Hodges Test (MHT) for the phenotypic detection of MBL production in clinical isolates.     

From Fig. 3: The test organism is E. coli strains (Nos. 82, 81, 89 and 51) producing metallo-β-lactamase (MBL) enzyme phenotypically. Strain No. 81 expressed MBL phenotypically, and this is noted by the inability of the test antibiotic (imipenem-10 µg) to inhibit the growth of the organism. MHT is performed by inoculating a Mueller-Hinton (MH) agar plate with a suspension of E. coli ATCC 25922 (adjusted to 0.5 McFarland turbidity standards) as per the CLSI criteria. A carbapenem disk (e.g. imipenem-10 10 µg) is aseptically placed at the center of the MH agar inoculated plate. A sterilized inoculating loop is used to streak the test bacteria (from an overnight culture) onto the MH agar plate in a straight line as shown in this illustration. The test organism should be heavily streaked (i.e. about 3-5 colonies should be picked), and the presence of growth of the test bacteria towards the carbapenem disk (arrows) is indicative of MBL production phenotypically. The test result is reported as positive in this case. 


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