Hodge Test Positive: Introduction, Principle, Procedure and Result Interpretation

Modified Hodge test Positive

Modified Hodge Test positive

Modified Hodge Test (MHT) is a simple and effective phenotypic test for the detection of the presence of carbapenemase enzyme in bacteria. A positive MHT test is common in Klebsiella pneumoniae carbapenemase (KPC), Metallo beta-lactamase (MBL), and the SME-1 in Serratia marcescens.
A clover leaf-like indentation of the E.coli 25922 growing along with the test organism growth streak within the disk diffusion zone indicating modified Hodge test Positive. Lacking growth of the E.coli 25922 along the test organism growth streak within the disc diffusion zone showing MHT negative.

Importance of Modified Hodge Test (MHT)

This test is useful for the following reasons-

  1. Earlier Carbapenem was an effective drug for treatment but nowadays it is becoming ineffective mostly due to the emergence of carbapenemase.
  2. This type of resistance among bacterial isolates is the leading cause of increased mortality and morbidity worldwide.
  3. To provide practical suggestions for clinicians dealing with the management of infections in critically ill patients, based on expert opinions.
  4. It is applicable in various Gram-negative bacilli (bacteria) like  E. coli, Pseudomonas aeruginosa , Klebsiella pneumoniae, Acinetobacter baumannii , Citrobacter diversus, Enterobacter agglomerans, etc.
  5. Modified Hodge test is very useful to avoid treatment failures and the development of resistance due to unnecessary use of this class of antibiotics.
  6. This test is a useful tool for ruling out Klebsiella pneumoniae carbapenemase.

Principle

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.

 Test Requirements

  • 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)
  • Others accessories
  • Saline, swab stick, forceps, inoculating loop, Bunsen burner,0.5 McFarland densitometer, or Standard

Procedure 

  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 37°C in ambient air for 16–24 hours.

Result and Interpretation

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. 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. e.g. Negative Control

Modifications of Hodge Test 

  • Hodge Test: Original Hodge test uses to evaluate the PCR confirmed IMP-1 and VIM-2 Metallo β-lactamase (MBL) producing isolates. The use of an imipenem 10µg disc gives fairly good results in MBL producing Pseudomonas aeruginosa and Acinetobacter spp.
  • Modified Hodge Test (MHT): As shown above
  • Triton Hodge Test (THT): It confers a large improvement to the sensitivity of the MHT for the detection of NDM and other carbapenemases.

Betalactamase detection|ESBL| MBL| KPC |AmpC |Oxacillinase|MRSA|D Zone test positive| iMLSB strain as shown in video-

Beta-lactamase detection: MRSA detection: D-Zone test: Positive/ iMLSB strain Screening and confirmatory tests for ESBL/Extended Spectrum beta Lactamase test MBL/Metallo beta-lactamase Ampicillinase /AmpC test Oxacillinase /OXA-48 test MRSA/ Methicillin-Resistant Staphylococcus aureus / MRSA D-Zone test for iMLSB strain detection Induced macrolide lincosamide and streptogramin

Bibliography

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3330182/
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521806/
  3. https://jcm.asm.org/content/54/3/640
  4. Bedenić B. [Beta-lactamases in laboratory and their role in resistance Part I.: Evolution of bacterial resistance mediated by beta-lactamases]. Lijecnicki vjesnik. 2003;126(11-12):314-24.
  5. Giedraitienė A, Vitkauskienė A, Naginienė R, Pavilonis A. Antibiotic resistance mechanisms of clinically important bacteria. Medicina (kaunas). 2011;47(3):137-46.
  6. Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clinical microbiology reviews. 2001;14(4):933-51.
  7. Samaha-Kfoury JN, Araj GF, Anwar R. Recent developments in [beta] lactamases and extended spectrum [beta] lactamases. British Medical Journal. 2003;327(7425):1209.
  8. Ahmed I, Salam A. Extended-spectrum beta-lactamases and bacterial resistance. PAKISTAN JOURNAL OF MEDICAL SCIENCES. 2002;18(2):151-5.
  9. Knothe H, Shah PDP, Krcmery V, Antal M, Mitsuhashi S. Transferable resistance to cefotaxime, cefoxitin, cefamandole, and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection. 1983;11(6):315-7.
  10. Jacoby GA, Medeiros AA. More extended-spectrum beta-lactamases. Antimicrobial Agents and Chemotherapy. 1991;35(9):1697.
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