Novobiocin is an antibiotic of the aminocoumarin family that is produced by the actinobacterium Streptomyces niveus and inhibits the production of bacterial DNA. Novobiocin suppresses DNA transcription by interacting with the DNA gyrase enzyme's GyrB subunit. Additionally, DNA topoisomerase IV activity is observed to be inhibited by it. The antibiotic was first suggested as a treatment for infections brought on by Gram-positive cocci. When other antibiotics proved ineffective for treating Staphylococcus aureus infections, it was authorised as a recommended therapy option in September 1964. However, the FDA banned its use as a therapeutic measure in 2009 due to its toxicity and diminished efficacy. Even though novobiocin isn't used for treatment, clinical laboratories still utilise it in the Novobiocin Susceptibility Test to identify and separate distinct strains of Staphylococcus. Novobiocin is susceptible to S. aureus and the majority of the coagulase-negative Staphylococci species described as pathogens, while S. saprophyticus is inherently resistant to it, according to research by Kloos and Schleifer from 1975. To distinguish S. saprophyticus from other CONS, they developed a novobiocin susceptibility test using this theory.
Table of Contents
- Objective
- Principle of Novobiocin Susceptibility Test
- Requirements
- Procedure of Novobiocin Susceptibility Test
- Result and Interpretation of Novobiocin Susceptibility Test
- Quality Control
- Precautions
- Applications of Novobiocin Susceptibility Test
- Limitations of Novobiocin Susceptibility Test
Objective
- To first and foremost distinguish Staphylococcus saprophyticus from other Coagulase Negative Staphylococci (CONS), particularly Staphylococcus epidermidis.
- Novobiocin-susceptible CONS and novobiocin-resistant CONS are divided into two categories.
Principle of Novobiocin Susceptibility Test
Novobiocin disrupts the synthesis of bacterial DNA, which prevents bacterial replication and colony growth. Novobiocin is effective against a variety of Staphylococcus species, including CONS and Coagulase Positive Staphylococci. The most prevalent Staphylococcaceae pathogens are S. aureus, S. saprophyticus, S. epidermidis, S. hominis, and S. haemolyticus. In the CONS group, S. saprophyticus and S. epidermidis are the most prevalent uropathogens. Except for S. saprophyticus, all of these pathogens are responsive to novobiocin. Based on this antimicrobial sensitivity pattern, we can quickly distinguish S. saprophyticus from other widespread CONS.
Requirements
Culture Media
The novobiocin susceptibility test is often carried out on Mueller Hinton Agar or Blood Agar. However, other forms of popular culture can also be utilised.
Preparation of MHA Plate
- According to the manufacturer's instructions, weigh the proper quantity of MHA powder (or the media components) and combine it with the necessary amount of water in a conical flask (or glass bottle).
- Stir thoroughly with a magnetic stirrer or by hand, then heat to boiling to completely dissolve the agar in the water.
- The flask or bottle should be autoclaved at 121°C and 15 lbs of pressure for 15 minutes, then cooled to between 40 and 45°C.
- Pour about 25 mL of the MHA into a sterile Petri dish (glass plate with a 10 cm diameter). (make around 4 mm thickness of the media in the petri plate).
- Keep the medium at room temperature to allow for full solidification. (Store in a freeze at 4°C for use up to 4 weeks)
Preparation of BA Plate (BAP)
- In a conical flask (or glass bottle), combine the requisite volume of water with the appropriate amount of Blood agar base powder (or the media components) in accordance with the manufacturer's instructions.
- Stir thoroughly with a magnetic stirrer or by hand, then heat to boiling to completely dissolve the agar in the water.
- The flask or bottle should be autoclaved at 121°C and 15 lbs of pressure for 15 minutes, then cooled to between 40 and 45°C.
- In the flask containing the blood agar base, slowly add 5% (5–10%) v/v sterile defibrinated blood (defibrinated sheep blood is preferable) while stirring continuously. Mix well so that the blood disintegrates into the medium equally. The Blood Agar is this concoction.
- Pour around 25 mL of the blood agar into a sterile Petri plate (a glass dish with a 10 cm diameter) and let it to harden properly by allowing it to sit at room temperature. (Store BAPs in a freeze at 4°C for use up to 2 to 4 weeks maximum)
Reagents
- Novobiocin Antibiotic Disk (5 μg)
- Blood from Defibrinated Sheep (for BAP preparation)
- Standard McFarland (1 McFarland and 0.5 McFarland suspension)
Composition
1% anhydrous barium chloride (BaCl2) solution
1% sulfuric acid (H2SO4) solution
Preparation of number 1 McFarland Standard Suspension
Add 9.9 mL of 1% H2SO4 and 0.1 mL of 1% BaCl2 solution to a clean, transparent test tube.
Preparation of number 0.5 McFarland Standard Suspension
Add 9.95 mL of 1% H2SO4 and 0.05 mL of 1% BaCl2 solution to a clean, transparent test tube.
Equipment
- Petri Plates
- Forceps
- Weighing Machine
- Autoclave
- Bunsen burner
- Sterile Test Tubes
- Micropipette
- Inoculating loop (Cotton Swab)
- PPE and other general laboratory materials.
Test bacteria
- Gram-positive, catalase-positive cocci in a cluster or short chain (Staphylococcus spp.)
- Staphylococcus aureus ATCC 25923
- Staphylococcus saprophyticus ATCC 15305
Procedure of Novobiocin Susceptibility Test
Both the Hebert technique, which uses BAP, and the Kirby Bauer Disk Diffusion technique, which uses an MHA plate, may be used to do this test.
1. Make a test bacterial suspension in a sterile test tube with sterile distilled water (or tryptic soy broth) and turbidity equivalent to McFarland number 1 if you are using BAP. If you're using MHA, prepare a bacterial suspension in a sterile test tube with sterile distilled water (or tryptic soy broth) that has a turbidity of 0.5 McFarland units.
2. The bacterial solution should be inoculated in agar plates using either a cotton swab or an inoculating loop.
- For BAP, streak/spread one area of the plate in one direction to inoculate.
- In MHA, inoculate by streaking/spreading in three directions (uniformly all over the plate)
3. Allow the suspension to stick to the plate and dry while keeping it upright for five to ten minutes.
4. A novobiocin antibiotic disc should be applied to the site of inoculation using sterile forceps, and it should be gently pressed to achieve adhesion.
5. Overnight, incubate the infected plate in an aerobic environment at 35 °C. (18 hours for MHA and 24 hours for BAP).
6. After the incubation time, watch for the development of a zone of inhibition around the antibiotic disc novobiocin, and then gauge the zone's diameter.
Result and Interpretation of Novobiocin Susceptibility Test
For BAP (Hebert method)
- Zone size of ≥12 mm = Sensitive to novobiocin
- Zone size of <12 mm = Resistant to novobiocin
For MHA (Kirby Bauer Disk Diffusion Method) (According to CLSI)
- Zone size of >16 mm = Sensitive to novobiocin
- Zone size of ≤16 mm = Resistant to novobiocin
If the test Staphylococcus spp. is isolated from a urine sample and it is coagulase-negative and novobiocin resistant, report it as Staphylococcus saprophyticus.
(However, it doesn’t confirm the bacterium as S. saprophyticus and must not be used if the bacteria are isolated from any samples other than urine.)
Quality Control
S. aureus ATCC 25923 (sensitive to novobiocin), which produces an antibiotic-resistant zone of around 22 mm around the novobiocin disk, is utilised as a positive control.
S. saprophyticus ATCC 15305 (resistant to novobiocin) is utilised as a negative control, developing an antibiotic resistant zone of 15 mm around the novobiocin disc.
Novobiocin Susceptible (Sensitive) Staphylococci
S. aureus, S. epidermidis, S. haemolyticus, S. hominis, S. capitis, S. saccharolyticus, etc.
Novobiocin Resistant Staphylococci
S. saprophyticus, S. kloosii, S. cohnii, etc.
Precautions
- Work in a sterile location, adequately sterilise the medium before use, wear the appropriate PPE, and abide by all laboratory safety regulations.
- If the temperature is beyond 45°C, avoid pouring blood onto molten blood agar foundation.
- Before the molten media's temperature falls below 40°C, pour the media into petri plates in a sterile area to prevent clumps from forming.
- When pouring the medium, make sure there are no air bubbles. Place the plates in a sterile area (such as the biosafety cabinet) where they can solidify. Leave the lid of the plate slightly ajar so that vapour may escape and water drops won't fall on the solidified media.
- Before using, let the agar plates fully firm. To guarantee complete solidification, prepare it at least 6 hours (or 1 day) before usage. Never put media in the refrigerator to solidify; always let it do so at room temperature.
- Place the disc 25 mm or so from the plate's edge. If using more than one antibiotic disk, space them apart by at least 25 mm.
Applications of Novobiocin Susceptibility Test
- For the purpose of distinguishing S. saprophyticus from other pathogenic CONS.
- During urine culture, distinguishing S. epidermidis and S. saprophyticus.
Limitations of Novobiocin Susceptibility Test
- S. saprophyticus can only be identified using this technique as a confirming one if the bacteria are isolated from a urine sample.
- To find the bacteria, additional biochemical tests are required.
- Due to the fact that it is a culture-based process, it takes longer and uses various ingredients and needs.
- A false positive or false negative result has a high probability.
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