Modified Hodge Test for Carbapenemase Detection: Principle, Procedure and Result Interpretations

Principle of Modified Hodge Test 

Modified Hodge Test (MHT) is a simple phenotypic test for the detection of the presence of carbapenemase enzyme in bacteria.  A positive MHT test is observed most commonly in Klebsiella pneumoniae carbapenemase (KPC), Metallo beta-lactamase (MBL), and the SME-1 in Serratia marcescens. Modified Hodge Test (MHT) has been suggested as screening tests for carbapenemases.

It is based on the inactivation of a carbapenem by carbapenemase-producing strains i.e.test isolate that enables a carbapenem -susceptible indicator strain (E. coli ATCC25922) to extend growth towards a carbapenem-containing disc along with the streak of inoculum of the test strain. A positive test result gives cloverleaf-like indentation.

 Requirements For Modified Hodge Test 

• Mueller -Hinton Agar (MHA)
• Bacteria:

Escherichia coli ATCC 25922 (indicator organism)

Positive Control (Klebsiella pneumoniae ATCC BAA-1705)

Negative Control (Klebsiella pneumoniae ATCC BAA-1706)

• Test strains (carbapenem-resistant)
• Other accessories: Saline, swab stick, forceps, inoculating loop, Bunsen burner,0.5 McFarland densitometer, or Standard

Procedure of Modified Hodge Test 

1. Prepare a 0.5 McFarland dilution of the E.coli ATCC 25922 (indicator organism) in 5 ml of saline.
2. Dilute 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of saline.
3. Streak a lawn of the 1:10 dilution of E.coli ATCC 25922 to a Mueller Hinton agar (MHA) plate and allow to dry 3–5 minutes.
4. Place a 10 µg meropenem or ertapenem susceptibility disc in the center of the test area.
5. In a straight line, streak test organism from the edge of the disc to the edge of the plate. Repeat the same with the QC strain in another direction.
6. Note: Up to four organisms can be tested on the same plate with one drug but we have used three as shown in the figure.
7. Incubate overnight at 37⁰C in ambient air for 16–24 hours.

Result and Interpretation of Modified Hodge Test 

After over incubation, examine the plate for a clover leaf-type indentation at the intersection of the test organism and the E. coli 25922, within the zone of inhibition of the carbapenem susceptibility disc.

• MHT Positive: It has a clover leaf-like indentation of the E.coli 25922 growing along with the test organism growth streak within the disc diffusion zone.
• A positive test indicates carbapenemase production by the test organism. By
• producing carbapenemase, the test microorganism is able to inactivate the carbapenem that diffuses from the disc after the disc has been placed on the MHA. This allows carbapenem susceptible E. coli ATCC 25922  to grow toward the disc. For e.g. Positive Control and Test
• MHT Negative: It has no growth of the E.coli 25922 along with the test organism growth streak within the disc diffusion zone. For e.g. Negative Control

Further Readings

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521806/
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3330182/
3. https://clsi.org/media/1899/_m100_archived_methods_table.pdf
4. https://journals.asm.org/doi/10.1128/JCM.00193-17
5. https://www.hindawi.com/journals/bmri/2014/139305/
6. https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-016-0754-z