The modified Hodges test or Cloverleaf test is another phenotypic test that is used to phenotypically confirm metallo beta lactamase (MBL) production in a test bacterial isolate after carrying out the screening test. This test is usually performed by aseptically swabbing Mueller-Hinton (MH) agar plates with Escherichia coli ATCC 25922 strain.
The inoculated MH agar plates should be allowed for about 5 min; and imipenem (10 µg), meropenem (10 µg) and ertapenem (10 µg) laden single disks should be aseptically placed 15 mm apart on the MH agar plates.
The test bacteria which showed reduced susceptibility or resistance to any of the carbapenems used in the screening test, and which have already been adjusted to 0.5 McFarland turbidity standards should be heavily streaked in three different directions from the edge of each of the disk to the center of the MH agar plates.
The MH agar plates should be incubated for 18-24 hrs at 37oC. The plates should also be macroscopically observed for indentation, and the growth of the test bacteria towards any of the carbapenem (imipenem, meropenem and ertapenem) susceptibility disk. Growth of test bacteria towards the carbapenem disk is indicative of 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):
- 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.