In vitro cost-effective methods to detect carbapenemases in Enterobacteriaceae

Carba NP test

Materials and reagents required

• Clinical laboratory reagent water (CLRW)

• Imipenem reference standard powder (Sigma-Aldrich Chemie GmbH 10160, Germany)

• Commercially available B-PER bacterial protein extraction reagent in Tris-HCl buffer, pH 7.4 Product #78248 (Thermo Scientific, 3747 N. Meridian Road, Rockford, IL 61101 USA)

• Zinc sulfate heptahydrate (Hi Media Laboratories Pvt. Ltd. Works, Mumbai, India)

• Phenol red powder (Hi Media Laboratories Pvt. Ltd. Works, Mumbai, India)

• 1N NaOH solution

• 10% HCl solution

• Microcentrifuge tubes 1.5 ml, clear

• 1 μl inoculation loops

• Containers to store prepared solutions.

Preparation of reagents

• 10 mM zinc sulfate heptahydrate solution:

  • Weigh out 1.4 g ZnSO₄7H₂O

  • Add to 500 ml CLRW

  • Mix

  • Store at room temperature.

Expiration: 1 year or not to exceed expiration of individual component.

• 0.5% phenol red solution:

  • Weigh out 1.25 g phenol red powder

  • Add to 250 ml CLRW

  • Mix

  • Store at room temperature.

Expiration: 1 year or not to exceed expiry of individual components.

Note: This solution does not remain in solution. Mix well before use.

• 0.1N sodium hydroxide solution:

  • Add 20 ml 1 N NaOH to 180 ml CLRW

  • Store at room temperature.

Expiration: 1 year or not to exceed expiry of individual components.

• Carba NP solution A:

  • In a 25–50 ml beaker, add 2 ml 0.5% phenol red solution to 16.6 ml CLRW

  • Add 180 μl of 10 mM zinc sulfate solution

  • Adjust pH to 7.8 ± 0.1 with 0.1 N NaOH solution if pH is too high

  • Store at 4–8°C in small vial or bottle and protect from prolonged light exposure.

Expiration: 2 weeks or not to exceed expiration of individual components (solution should remain red or red-orange; do not use if solution turns any other color).

• Carba NP solution B (solution A + 6 mg/ml imipenem powder):

  • Determine the amount of solution B needed, allowing 100 μl per tube for each patient, quality control (QC) strain, and an uninoculated reagent control

  • Weigh out approximately 10–20 mg of imipenem powder.

Note: it is advisable to weigh out at least 10 mg of powder. Suppose we have weighed 12 mg powder, divide the actual weight by 6 to determine the amount (in ml) of solution A to be added to the powder. As the concentration required for test is 6 mg/ml.

Example: 12 mg of imipenem/6 = 2 ml of solution A, which is sufficient for 20 tubes.

• Store at 4–8°C for up to 3 days.

Quality control

Control strains included for routine laboratory carbapenem susceptible strains are used to rule out or minimize false positivity and to evaluate the specificity of the test methodology.

QC strains are also used as:

• K. pneumoniae ATCC BAA-1705-MHT positive

• K. pneumoniae ATCC BAA-1706-MHT negative.

Test procedure

1. Label two microcentrifuge tubes (one “a” and one “b”) for each patient isolate, QC organism, and uninoculated reagent control

2. Add 100 μl of bacterial protein extraction reagent to each tube

3. For each isolate to be tested, emulsify a 1 μl loopful of bacteria from an overnight growth culture in both tubes “a” and “b”

4. Vortex each tube for 5 s (uninoculated reagent control tubes should contain only bacterial protein extraction reagent, no organism)

5. Add 100 μl of Solution A to tube “a”

6. Add 100 μl of solution B to tube “b”

7. Vortex tubes well

8. Incubate at 35°C ± 2°C for up to 2 h

9. Isolate that demonstrate positive results before 2 h can be reported as carbapenemase producers.

Note: Do not use growth from selective media or plates containing antibiotics or other agents that select for certain bacteria.

Test interpretation

1. Read uninoculated reagent control tubes “a” and “b”

   • Both tubes must be red or red-orange.

2. Read inoculated tube “a”

   • Tube “a” must be red or red-orange

   • If tube “a” is any other color, test is invalid.

3. Read inoculated tube “b”

   • Red or red-orange = negative

   • Light orange, dark yellow or yellow = positive

   • Orange = invalid [Figures 1 and 2].

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Figure 1

Inoculated tubes (a) showing red color

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Figure 2

Inoculated tubes (b) showing yellow color positive for carbapenemase production

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Reporting

Report positive if color changes From red color to yellow color in test tube.

Modified Carba NP test

Materials and reagent required

1. In MCNP method, the therapeutic intravenous (i.v.) imipenem/cilastatin with imipenem IP equivalent to 500 mg powder is used which is used commercially for patient use

2. Lysis buffer used is 0.02% cetyltrimethylammonium bromide (CTAB - Sigma-Aldrich Chemie, GmbH, Germany)

3. pH should be below 7.8

4. No centrifugation and incubation required.

In house modification of the method

In MCNP method, the original publication used imipenem/cilastatin powder with concentration of 6 mg/ml and pH <7.8; however, in our modification, we have increased the concentration of imipenem/cilastatin powder to 8 mg/ml and pH is adjusted to 7.5.

Test procedure

1. Label two tubes for one strain tube “a” and tube “b”

2. Tube “a” as test tube and tube “b” as negative control tube

3. Add 200 μl of 0.02% CTAB (lysis buffer) to each tube

4. Add 100 μl bacterial suspension to each tube

5. Add 100 μl diluted phenol red solution, pH <7.5 to negative control tube “b”

6. Add 100 μl diluted phenol red solution supplemented with imipenem/cilastatin (concentration 8mg/ml) in tube “a”.

7. Vortex tube “a” and tube “b”

8. Incubate at 37°C for 2 h.

Test interpretation

Test tube “a” versus negative control tube “b” and yellow-orange color versus red color is considered as positive for carbapenemases production.

Note: If color does not change to yellow or orange, it should be incubated for 2 h to declare it negative. If color still remains red, then label it as negative for the test [Figure 3].

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Figure 3

Five red color tubes (b) as ‘control’ and next five yellow color tubes (a) as ‘positive’ for carbapenemase production

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Carbapenem inactivation method

Material and reagents required

1. Mueller-Hinton agar (MHA) or blood agar plates

2. Distilled water

3. 10 μg of meropenem disk (Hi Media Laboratories Pvt. Ltd. Works, Mumbai, India)

4. Control strain of E. coli ATCC – 25922.

Test procedure

1. Add 10 μl inoculation loopful of culture of tested strain in 400 μl distilled water

2. Put 10 μg of meropenem disk into suspension

3. Incubate it for 2 h at 35°C

4. Treated meropenem disk is placed on MHA plates inoculated with ATCC E. coli – 25922 strain as lawn culture

5. Incubate it for 8–10 hours at 37°C [Figure 4].

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Figure 4

Carbapenem inactivation method, showing test strain was resistant on treated meropenem disk, interpreted as positive for carbapenemase production

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Interpretation of result

1. If test strain is meropenem resistant, i.e., no zone of inhibition – positive for carbapenemase production and meropenem is hydrolyzed

2. If test strain is sensitive to meropenem – negative for carbapenemase production showing zone of inhibition by meropenem.

Rapidec Carba NP kit test

Material and reagents required

1. Rapidec test kit (BioMerieux SA, France)

2. Test strains

3. 10–100 μl pipette

4. Sterile tips.

Test procedure as suggested by manufacturers

1. Step 1: Add 100 μl suspension given with kit in well “a,” “b,” “c” for rehydration of well for 5–10 min with lid covered at room temperature

2. Step 2: Mix the content of well “b” with stirrer

3. Step 3: Add bacterial colonies into well “c” with same turbidity of well “b” and then cover with lid for 30 min at room temperature

4. Step 4: Transfer 25 μl from well “c” to both well “d” and well “e”(well “d” is control well and well “e” is test well)

5. Step 5: Transfer 25 μl from well “a” to both well “d” and well “e”

6. Step 6: Cover the lid and incubate it for 30 min at 37°C [Figure 5].

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Figure 5

Rapidec Carba NP test kit, depicting red color in well “d” and yellow color in well “e,” interpreted as positive for carbapenemase production

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Test interpretation

1. First reading should be taken after 30 min. If positive for carbapenemase production, well “d” changes to red color and well “e” yellow to orange color

2. For negative result second reading taken after 30 min.