Modified Hodge Test (MHT)- Principle, Procedure, Results, Uses

Carbapenems are a class of broad-spectrum β-lactam antibiotics that are effective against many Gram-negative and Gram-positive bacteria. They are often used as the last resort for treating infections caused by multidrug-resistant organisms. However, some bacteria have developed resistance to carbapenems by producing enzymes called carbapenemases, which can hydrolyze and inactivate these antibiotics. Carbapenemase-producing organisms (CPOs) pose a serious threat to public health as they can spread rapidly and cause outbreaks of infections that are difficult to treat.

Therefore, it is important to detect CPOs in clinical specimens and monitor their prevalence and epidemiology. There are different methods for detecting carbapenemase production, such as molecular methods, biochemical methods, and phenotypic methods. Molecular methods involve detecting the genes encoding carbapenemases by PCR or sequencing. Biochemical methods involve detecting the enzymatic activity of carbapenemases by using specific substrates or inhibitors. Phenotypic methods involve detecting the phenotypic expression of carbapenemase production by using antibiotic susceptibility testing or other indicators.

Modified Hodge Test (MHT) is one of the phenotypic methods for detecting carbapenemase production. It was first described by Lee et al. in 2001 and later modified by the Clinical and Laboratory Standards Institute (CLSI) in 2009. It is based on the principle that the carbapenemase enzyme produced by the test organism hydrolyzes the carbapenem antibiotic resulting in the enhanced growth of carbapenem-susceptible E. coli. The test involves inoculating a Mueller Hinton Agar (MHA) plate with E. coli and placing a carbapenem disc at the center. Then, the test organism is streaked from the edge of the disc to the edge of the plate. After incubation, if the test organism produces carbapenemase, it will enhance the growth of E. coli within the zone of inhibition of the disc, forming a clover leaf-like pattern.

MHT is a simple and inexpensive method that can be performed in most laboratories without requiring specialized equipment or reagents. However, it also has some limitations, such as false positive results due to other β-lactamase enzymes, false negative results due to low-level carbapenemase production, and interference from some metabolites produced by the test organism. Therefore, MHT should be used in conjunction with other methods for confirming carbapenemase production.

In this article, we will discuss the objectives, principle, requirements, procedure, observation, result interpretation, advantages and limitations of MHT in detail.

The main objective of Modified Hodge Test (MHT) is to detect carbapenemase production by bacteria. Carbapenemases are enzymes that can hydrolyze carbapenems, which are a class of broad-spectrum antibiotics that are usually effective against multidrug-resistant Gram-negative bacteria. Carbapenemase-producing bacteria are a serious threat to public health as they can cause infections that are difficult to treat and can spread rapidly in healthcare settings.

Another objective of Modified Hodge Test (MHT) is to distinguish between carbapenemase-mediated resistance and other mechanisms of carbapenem resistance. Some bacteria may be resistant to carbapenems due to other factors, such as reduced permeability, efflux pumps, or production of other β-lactamases. These mechanisms may not confer resistance to all carbapenems or may be overcome by using higher doses or combinations of antibiotics. Therefore, it is important to identify the exact mechanism of resistance to guide appropriate therapy and infection control measures.

The Modified Hodge Test (MHT) is based on the principle that the carbapenemase enzyme produced by the test organism hydrolyses the carbapenem antibiotic resulting in the enhanced growth of carbapenem-susceptible E. coli.

Carbapenems are a class of broad-spectrum β-lactam antibiotics that are resistant to most β-lactamases. However, some bacteria can produce carbapenemases, which are enzymes that can inactivate carbapenems by breaking their β-lactam ring. Carbapenemases are usually encoded by genes located on plasmids, which can be transferred between different bacteria and confer resistance to multiple antibiotics.

The MHT uses a carbapenem-sensitive E. coli strain as an indicator organism and a carbapenem disc as a substrate. The test organism and the control strains are streaked from the edge of the disc to the edge of the plate on a Mueller Hinton Agar (MHA) plate inoculated with the E. coli strain. The plate is then incubated for 16-24 hours at 35°C.

If the test organism produces any carbapenemase enzyme, it will hydrolyze the diffused antibiotic in the vicinity of the streaked lines. This will enhance the growth of E. coli, which will otherwise be inhibited by the antibiotic, in the vicinity of the streak. This growth enhancement results in indented growth of the E. coli and test organism within the zone of inhibition of the E. coli giving a clover leaf-like pattern.

The MHT is a phenotypic method that detects the functional activity of carbapenemases rather than their genetic presence. It can be used to screen for carbapenemase-producing organisms among Gram-negative bacilli such as Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp.

To perform the Modified Hodge Test (MHT), you will need the following materials and equipment:

• Carbapenem sensitive E. coli (E. coli ATCC 25922 strain): This is the indicator organism that will show enhanced growth in the presence of carbapenemase-producing bacteria. You can obtain this strain from a reference laboratory or a commercial supplier.
• Mueller Hinton Agar (MHA) Plates: These are the culture media that will support the growth of E. coli and the test bacteria. You can prepare MHA plates by following the manufacturer`s instructions or using a standard protocol. Make sure to use freshly prepared or properly stored MHA plates for optimal results.
• Test bacteria: These are the bacteria that you want to test for carbapenemase production. You can use any clinical isolate or reference strain that is suspected or confirmed to be resistant to carbapenems. You should use freshly cultured bacteria from an overnight growth on a suitable medium.
• Control Bacteria: These are the bacteria that will serve as positive and negative controls for the MHT. You should use two control strains: one that is known to produce carbapenemase (such as Klebsiella pneumoniae ATCC BAA – 1705) and one that is known not to produce carbapenemase (such as Klebsiella pneumoniae ATCC BAA – 1706). You can obtain these strains from a reference laboratory or a commercial supplier. You should also use freshly cultured bacteria from an overnight growth on a suitable medium.
• Carbapenem antibiotic disc (10 mcg Etrapenem or Meropenem disc): This is the disc that will contain the carbapenem antibiotic that will be hydrolyzed by the carbapenemase enzyme. You can use either Etrapenem or Meropenem as the carbapenem antibiotic, depending on your preference and availability. You can obtain these discs from a commercial supplier or prepare them by using a standard protocol. Make sure to store them properly and use them before their expiry date.
• Cotton swab or bent glass rod (Dolly rod) and inoculating loop: These are the tools that you will use to inoculate and streak the bacteria on the MHA plates. You should use sterile cotton swabs or bent glass rods to spread the E. coli suspension on the MHA plates. You should use sterile inoculating loops to pick and streak the test and control bacteria on the MHA plates.
• Other general requirements: These include a Bunsen burner, sterile saline water (or broth), 0.5 McFarland standards, forceps, spirit, etc. You should use these materials and equipment to prepare and sterilize your tools and solutions, adjust the turbidity of your bacterial suspensions, and handle your culture plates safely and properly.

These are the basic requirements for performing the Modified Hodge Test (MHT). You should follow the standard operating procedures and biosafety guidelines when working with these materials and equipment. You should also label your culture plates clearly and record your observations carefully.

The Modified Hodge Test (MHT) is performed as follows:

1. Prepare a 0.5 McFarland standard suspension of E. coli ATCC 25922 (or E. coli strain sensitive to carbapenem) in sterile saline or broth.
2. Dilute the suspension with sterile saline or broth in the ratio of 1:10 (add 0.5 mL of suspension to 4.5 mL of saline or broth).
3. Uniformly streak or spread the diluted E. coli suspension on a sterile Mueller Hinton Agar (MHA) plate (like during the usual antimicrobial sensitivity test) and allow it to dry for about 3 – 10 minutes.
4. Place a disc of 10 mcg Meropenem or Etrapenem at the center of the inoculated MHA plate.
5. Using a sterile inoculating loop or swab, pick 3-5 colonies of freshly cultured test organisms and streak inside out in a straight line from the edge of the disc to the edge of the plate.
6. Incubate the plate at 35(±2)°C for 16 – 24 hours at ambient air.

After the incubation, observe the MHA plate for the presence or absence of enhanced growth of E. coli and formation of clover leaf-like indentation at the intersection of the streak of the test organism (and controls, if used) and the zone of inhibition of E. coli.

The enhanced growth of E. coli indicates that the test organism produces carbapenemase enzyme that hydrolyzes the carbapenem antibiotic and allows E. coli to grow in its vicinity. The clover leaf-like indentation is formed due to the indented growth of E. coli and test organism within the zone of inhibition of E. coli.

The absence of enhanced growth of E. coli indicates that the test organism does not produce carbapenemase enzyme or produces it in a very low amount that does not affect the carbapenem antibiotic and its inhibitory effect on E. coli.

The following image shows an example of a positive and a negative result of MHT:

A positive result is seen with K. pneumoniae ATCC BAA – 1705 (MHT positive control) as there is a clear clover leaf-like indentation at the intersection of its streak and the zone of inhibition of E. coli.

A negative result is seen with K. pneumoniae ATCC BAA – 1706 (MHT negative control) as there is no enhanced growth or indentation at the intersection of its streak and the zone of inhibition of E. coli.

The test organism (Pseudomonas aeruginosa) also shows a negative result as there is no enhanced growth or indentation at the intersection of its streak and the zone of inhibition of E. coli.

The result of the Modified Hodge Test (MHT) is based on the observation of the enhanced growth of E. coli and the formation of a clover leaf-like indentation at the intersection of the streak of the test organism and the zone of inhibition of E. coli. The enhanced growth indicates that the test organism produces carbapenemase enzyme that hydrolyzes the carbapenem antibiotic and allows E. coli to grow in its vicinity. The clover leaf-like indentation is a characteristic feature of MHT that distinguishes it from other phenotypic methods.

The result interpretation of MHT is as follows:

• Positive Result: If there is enhanced growth of E. coli and a clover leaf-like indentation at the intersection of the streak of the test organism and the zone of inhibition of E. coli, then the result is positive for carbapenemase production. This means that the test organism is resistant to carbapenem antibiotics due to carbapenemase production.

• Negative Result: If there is no enhanced growth of E. coli and no clover leaf-like indentation at the intersection of the streak of the test organism and the zone of inhibition of E. coli, then the result is negative for carbapenemase production. This means that the test organism is either susceptible to carbapenem antibiotics or resistant due to other mechanisms.

• Control Strains: The control strains are used to validate the performance of MHT and to ensure that the test conditions are optimal. The positive control strain (K. pneumoniae ATCC BAA – 1705) should show a positive result, while the negative control strain (K. pneumoniae ATCC BAA – 1706) should show a negative result. If the control strains do not show the expected results, then the test is invalid and should be repeated.

The following table summarizes the result interpretation of MHT:

Here is an example image showing positive and negative results of MHT:

The Modified Hodge Test (MHT) is a phenotypic method for detecting carbapenemase production in bacteria. It has some advantages and limitations that should be considered before using it as a diagnostic tool.

Advantages

• It is simple and easy to perform. It does not require specialized equipment or skills, and can be done with readily available materials such as Mueller Hinton Agar plates, E. coli strain, carbapenem disc, and inoculating loop.
• It is inexpensive and cost-effective. It does not involve expensive reagents or kits, and can be implemented by smaller laboratories and institutes with limited resources.
• It can detect different types of carbapenemases, such as KPC, NDM, OXA-48, and IMP. It can also detect some metallo-beta-lactamases (MBLs), such as VIM and NDM.

Limitations

• It is not very sensitive or specific. It can give false positive results due to the presence of other beta-lactamases, such as extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases, that can hydrolyze carbapenems. It can also give false negative results due to the low level of carbapenemase production or the presence of other resistance mechanisms, such as efflux pumps or porin loss.
• It is not standardized or validated. There is no consensus on the optimal conditions or criteria for performing and interpreting the MHT. Different protocols may use different E. coli strains, carbapenem discs, inoculum sizes, incubation times, and reading methods. This can lead to variability and inconsistency in the results.
• It is not suitable for all bacteria. Some bacteria may produce metabolites that inhibit the growth of E. coli, such as Pseudomonas aeruginosa or Acinetobacter baumannii. Some bacteria may grow poorly on Mueller Hinton Agar plates, such as anaerobes or fastidious organisms. Some bacteria may have intrinsic resistance to carbapenems, such as Stenotrophomonas maltophilia or Burkholderia cepacia.

Therefore, the MHT should be used with caution and in conjunction with other methods, such as molecular tests or phenotypic confirmatory tests, to accurately detect carbapenemase production in bacteria.

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