METALLO-β-LACTAMASE (MBL)

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Metallo-beta-lactamases (MBLs) are beta-lactamases 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. Their presence in clinically important Gram-negative bacteria has 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 such as 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 in vitro.

Genes responsible for the expression of MBLs in pathogenic Gram-negative bacteria can also be chromosomally or plasmid-mediated as is also applicable with 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 such as ESBL positive bacteria. The carbapenems are very potent antimicrobial agents used for the treatment of serious Gram-negative bacterial infections. Because of the broad-spectrum activity and stability of the carbapenems to most beta-lactamase, they have been widely used under restricted conditions in most hospitals as the first-line treatment for severe Gram-negative infections. MBLs are mainly expressed by non-lactose fermentors 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 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. 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. 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 and confirmatory tests (Figure 1).

Figure 1. Screening of a clinical isolateof P. aeruginosa for susceptibilty to imipenem (10 µg) and meropenem (10 µg). The P. aeruginosa isolate is resistant (R) to the imipenem and meropenem antibiotics (Oxoid, UK). Reduced susceptibility 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. Photo courtesy: https://www.microbiologyclass.com

PCR technique can also be applied 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 is first evaluated by the Kirby-Bauer disk diffusion test. Organisms found to be resistant to any of these agents (based on the breakpoints of the Clinical and Laboratory Standard Institute, CLSI) are subjected to a phenotypic confirmation test for MBL detection. The disk potentiation or inhibitor-based assays (Figure 2) and the modified Hodges test (Figure 3) are usually used for the phenotypic confirmation of MBL production in bacteria.

Figure 2. Disk potentiation/Inhibitor based assay for the phenotypic confirmation of MBL production in clinical isolates. IPM=imipenem (10 µg); MEM=meropenem (10µg); EDTA=ethylenediamine tetraacetic acid. 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 when used alone and in combination with each imipenem and meropenem disks containing 0.5 µl EDTA infers MBL production phenotypically. Photo courtesy: https://www.microbiologyclass.com
Figure 3. Modified Hodges Test (MHT) for the phenotypic detection of MBL production in clinical isolates. 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 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. Photo courtesy: https://www.microbiologyclass.com

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 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 isolates. 

FURTHER READING

Ashutosh Kar (2008). Pharmaceutical Microbiology, 1st edition. New Age International Publishers: New Delhi, India. 

Block S.S (2001). Disinfection, sterilization and preservation. 5th edition. Lippincott Williams & Wilkins, Philadelphia and London.

Courvalin P, Leclercq R and Rice L.B (2010). Antibiogram. ESKA Publishing, ASM Press, Canada.

Denyer S.P., Hodges N.A and Gorman S.P (2004). Hugo & Russell’s Pharmaceutical Microbiology. 7th ed. Blackwell Publishing Company, USA. Pp.152-172.

Ejikeugwu Chika, Iroha Ifeanyichukwu, Adikwu Michael and Esimone Charles (2013). Susceptibility and Detection of Extended Spectrum β-Lactamase Enzymes from Otitis Media Pathogens. American Journal of Infectious Diseases. 9(1):24-29.

Finch R.G, Greenwood D, Norrby R and Whitley R (2002). Antibiotic and chemotherapy, 8th edition. Churchill Livingstone, London and Edinburg.

Russell A.D and Chopra I (1996). Understanding antibacterial action and resistance. 2nd edition. Ellis Horwood Publishers, New York, USA.

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