Novobiocin Susceptibility Test- Principle, Procedure, Results

The main objective of the novobiocin susceptibility test is to differentiate Staphylococcus saprophyticus from other Coagulase Negative Staphylococci (CONS). This is important because S. saprophyticus is a common cause of urinary tract infections (UTIs), especially in young women, and it has a distinctive resistance to novobiocin, an antibiotic that inhibits bacterial DNA synthesis. By using a novobiocin disk on a culture medium, we can observe whether the test bacteria form a zone of inhibition (indicating susceptibility) or not (indicating resistance) around the disk.

Another objective of the test is to classify CONS into two groups based on their novobiocin sensitivity. CONS are a diverse group of bacteria that are usually harmless commensals on human skin and mucous membranes but can also cause opportunistic infections in immunocompromised patients or when they contaminate medical devices. Some of the most common CONS species are S. epidermidis, S. haemolyticus, S. hominis, and S. capitis. Most of these species are susceptible to novobiocin, except for some rare ones like S. kloosii and S. cohnii. By performing the novobiocin susceptibility test, we can narrow down the possible identification of CONS isolates and guide the appropriate antibiotic therapy.

Novobiocin is an antibiotic that interferes with the bacterial DNA synthesis process by inhibiting the activity of DNA gyrase, an enzyme that unwinds and supercoils the DNA strands. By blocking this enzyme, novobiocin prevents the replication and colony formation of bacteria.

Many of the Staphylococcus species, both coagulase-positive and coagulase-negative staphylococci (CPS and CNS), are susceptible to novobiocin. Among the pathogenic members of Staphylococcaceae, S. aureus, S. saprophyticus, S. epidermidis, S. hominis, and S. haemolyticus are the most common. S. saprophyticus and S. epidermidis are the most common uropathogens in the CNS group.

All these pathogens are sensitive to novobiocin except S. saprophyticus, which has a mutation in its DNA gyrase gene that confers resistance to novobiocin. This antimicrobial sensitivity pattern is the principle based on which we can easily differentiate S. saprophyticus from other common CNS.

By using a novobiocin antibiotic disk on a culture medium inoculated with the test bacteria, we can observe the formation of a zone of inhibition around the disk, which indicates the susceptibility or resistance of the bacteria to novobiocin. The size of the zone depends on the concentration of novobiocin in the disk, the diffusion rate of novobiocin in the medium, and the growth rate and density of the bacteria.

The zone size is measured in millimeters and compared with standard criteria to interpret the result as sensitive or resistant to novobiocin. A sensitive bacterium will have a larger zone of inhibition than a resistant bacterium.

If the test Staphylococcus spp. is isolated from a urine sample and it is coagulase-negative and novobiocin resistant, it is likely to be S. saprophyticus. However, this test does not confirm the identity of S. saprophyticus and must not be used if the bacteria are isolated from any samples other than urine. Other biochemical tests are required to confirm the identification of S. saprophyticus.

Culture Media

Generally, Mueller Hinton Agar (MHA) or Blood Agar (BA) is preferred for conducting the novobiocin susceptibility test. However, other general cultural media can also be used.

Preparation of MHA Plate

• Measure the appropriate amount of MHA powder (or the media components) and mix in the water of the required volume in a conical flask (or glass bottle) according to the instruction of the manufacturing company.

• Stir well using a magnetic stirrer or manually and heat to boiling so that all the components and agar dissolve completely in water.

• Autoclave the flask or bottle at 121°C and 15 lbs pressure for 15 minutes and let it cool to around 40 – 45°C.

• In a sterile Petri plate (glass plate with 10 cm diameter), pour around 25 mL of the MHA (make around 4 mm thickness of the media in the Petri plate).

• Let the media solidify completely by leaving it at room temperature. (Store in a freeze at 4°C for use up to 4 weeks)

Preparation of BA Plate (BAP)

• Measure the appropriate amount of Blood agar base powder (or the media components) and mix in the water of the required volume in a conical flask (or glass bottle) according to the instruction of the manufacturing company.

• Stir well using a magnetic stirrer or manually and heat to boiling so that all the components and agar dissolve completely in water.

• Autoclave the flask or bottle at 121°C and 15 lbs pressure for 15 minutes and let it cool to around 40 – 45°C.

• Pour 5% (5 to 10%) v/v sterile defibrinated blood (defibrinated Sheep Blood is preferred) in the flask with blood agar base slowly with constant stirring. Mix properly so that blood dissolves uniformly in the medium. This mixture is the Blood Agar.

• In a sterile Petri plate (glass plate with 10 cm diameter), pour around 25 mL of the blood agar and let it solidify properly by leaving it 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)

• Defibrinated Sheep Blood (for BAP preparation)

• McFarland Standard (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

• In a clean and clear test tube, add 9.9 mL of 1% H2SO4 and 0.1 mL of 1% BaCl2 solution

Preparation of number 0.5 McFarland Standard Suspension

• In a clean and clear test tube, add 9.95 mL of 1% H2SO4 and 0.05 mL of 1% BaCl2 solution

Equipment

PPE and other general laboratory materials.

Test bacteria

Gram-positive, catalase-positive cocci in a cluster or short chain (Staphylococcus spp.)

Staphylococcus aureus ATCC 25923Staphylococcus saprophyticus ATCC 15305

In general, this test can be performed using two slightly different methods; the Hebert method using BAP and Kirby Bauer Disk Diffusion Method using an MHA plate.

Hebert Method Using BAP

• Make a test bacterial suspension with sterile distilled water (or tryptic soya broth) in a sterile test tube with turbidity equal to McFarland number 1.

• Using a cotton swab or inoculating loop, inoculate the bacterial suspension in a BAP plate by streaking/spreading one section of the plate in one direction.

• Let the suspension adhere to the plate and dry, leaving the plate in an upright position for about 5 to 10 minutes.

• Using sterile forceps, place a novobiocin antibiotic disk (5 μg) in the area of inoculation and gently press the antibiotic disk to ensure its adherence.

• Incubate the inoculated plate aerobically at 35±2°C for 24 hours.

• Following the incubation period, observe the formation of a zone of inhibition around the novobiocin antibiotic disk and measure the zone diameter.

Kirby Bauer Disk Diffusion Method Using MHA Plate

• Make a test bacterial suspension with sterile distilled water (or tryptic soya broth) in a sterile test tube with turbidity equal to McFarland number 0.5.

• Using a cotton swab or inoculating loop, inoculate the bacterial suspension in an MHA plate by streaking/spreading in three directions (uniformly all over the plate).

• Let the suspension adhere to the plate and dry, leaving the plate in an upright position for about 5 to 10 minutes.

• Using sterile forceps, place a novobiocin antibiotic disk (5 μg) in the center of the plate and gently press the antibiotic disk to ensure its adherence.

• Incubate the inoculated plate aerobically at 35±2°C for 18 hours.

• Following the incubation period, observe the formation of a zone of inhibition around the novobiocin antibiotic disk and measure the zone diameter.

The result of the novobiocin susceptibility test is based on the measurement of the zone of inhibition around the novobiocin antibiotic disk. The zone of inhibition is the clear area where the bacterial growth is inhibited by the antibiotic. The size of the zone depends on the method used, the type of agar, and the concentration of the antibiotic disk.

For BAP (Hebert method), a zone size of ≥12 mm indicates that the test bacterium is sensitive to novobiocin, while a zone size of <12 mm indicates that it is resistant to novobiocin. For MHA (Kirby Bauer Disk Diffusion Method), a zone size of >16 mm indicates that the test bacterium is sensitive to novobiocin, while a zone size of ≤16 mm indicates that it is resistant to novobiocin.

If the test bacterium is coagulase-negative and isolated from a urine sample, and it shows resistance to novobiocin, it can be reported as Staphylococcus saprophyticus. This is because S. saprophyticus is the only common pathogenic CONS that is resistant to novobiocin. However, this test does not confirm the identity of S. saprophyticus and should not be used for bacteria isolated from samples other than urine.

The following table summarizes the result and interpretation of the novobiocin susceptibility test:

| Test Bacterium | Zone Size (BAP) | Zone Size (MHA) | Interpretation |

| -------------- | --------------- | --------------- | ------------- |

| S. aureus | ≥22 mm | ≥22 mm | Sensitive |

| S. epidermidis | ≥12 mm | >16 mm | Sensitive |

| S. saprophyticus| <12 mm | ≤16 mm | Resistant |

The following images show examples of positive and negative results of the novobiocin susceptibility test:

Positive result: S. aureus shows a large zone of inhibition around the novobiocin disk.

Negative result: S. saprophyticus shows no zone of inhibition around the novobiocin disk.

Quality control is an essential step in any laboratory test to ensure the accuracy and reliability of the results. For the novobiocin susceptibility test, quality control should be performed per lot/shipment date with known organisms that have predictable susceptibility patterns to novobiocin.

The positive control (resistant) for the novobiocin susceptibility test is Staphylococcus saprophyticus (ATCC® 15305), which should produce a zone of inhibition of less than 12 mm on BAP or less than or equal to 16 mm on MHA. The negative control (sensitive) for the novobiocin susceptibility test is Staphylococcus epidermidis (ATCC® 12228), which should produce a zone of inhibition greater than or equal to 16 mm on both BAP and MHA.

The quality control strains should be inoculated and tested in the same manner as the test strains, using the same media, reagents, and equipment. The quality control results should be recorded and compared with the expected results. If the quality control results are within the acceptable range, the test results can be reported. If the quality control results are outside the acceptable range, the test should be repeated or investigated for possible sources of error .

The novobiocin susceptibility test has two main applications in the clinical microbiology laboratory:

• For identification of S. saprophyticus among other pathogenic CONS. S. saprophyticus is a common cause of urinary tract infections (UTIs) in young women and men. It accounts for 10-20% of all UTIs and is second only to E. coli as the most frequent uropathogenic. S. saprophyticus is coagulase-negative and novobiocin-resistant, which distinguishes it from other CONS such as S. epidermidis, S. haemolyticus, and S. hominins, which are coagulase-negative and novobiocin-sensitive. Therefore, the novobiocin susceptibility test can help to confirm the presence of S. saprophyticus in urine samples and guide the appropriate antibiotic therapy.

• For differentiating S. epidermidis and S. saprophyticus during urine culture. S. epidermidis and S. saprophyticus are both coagulase-negative staphylococci that can colonize human skin and mucous membranes. They can also contaminate urine samples during collection or processing, leading to false-positive results and unnecessary treatment. To avoid this, urine samples should be cultured on selective media such as mannitol salt agar (MSA) or chromogenic agar that can differentiate staphylococci based on their ability to ferment mannitol or produce specific enzymes. However, these media cannot distinguish between S. epidermidis and S. saprophyticus, which are both mannitol-fermenting and produce similar colony colors on chromogenic agar. Therefore, the novobiocin susceptibility test can be used as an additional method to differentiate these two species based on their resistance or sensitivity to novobiocin.

The novobiocin susceptibility test is a simple, rapid, and inexpensive method that can provide valuable information for the identification and differentiation of coagulase-negative staphylococci in urine samples. It can help to diagnose UTIs caused by S. saprophyticus and avoid unnecessary treatment of urine contamination by S. epidermidis or other CONS.

The novobiocin susceptibility test is a simple and useful method to differentiate S. saprophyticus from other CONS, especially S. epidermidis, which are the most common uropathogens in this group. However, this test has some limitations that should be considered before using it as a diagnostic tool.

• The test can only be used as a confirmatory method for S. saprophyticus if the bacteria are isolated from a urine sample. This is because S. saprophyticus is mainly associated with urinary tract infections and rarely causes infections in other sites. Therefore, if the bacteria are isolated from other sources, such as blood, wound, or catheter, the test may not be reliable, and other identification methods should be used.

• The test does not provide definitive identification of the bacterium. It only indicates whether the bacterium is novobiocin susceptible or resistant. To confirm the identity of the bacterium, other biochemical tests, such as the coagulase test, mannitol fermentation test, urease test, and DNA sequencing, should be performed.

• The test is a culture-based method that requires a longer duration and different chemicals/requirements than molecular methods. The test involves preparing and inoculating agar plates, placing antibiotic disks, incubating overnight, and measuring zone sizes. This process may take up to 24 hours or more, depending on the growth rate of the bacteria and the availability of the materials. In contrast, molecular methods, such as PCR or MALDI-TOF MS, can provide rapid and accurate identification of bacteria within hours or minutes using DNA or protein analysis.

• The test has a high chance of producing false positive or false negative results due to various factors. Some factors that may affect the accuracy of the test are:

• The quality and concentration of the novobiocin antibiotic disk. The disk should contain 5 μg of novobiocin and should be stored properly to avoid degradation or contamination.

• The quality and thickness of the agar medium. The medium should be sterile and have a uniform thickness of about 4 mm. If the medium is too thin or too thick, it may affect the diffusion of the antibiotic and the formation of the zone of inhibition.

• The inoculum size and density of the bacteria. The bacterial suspension should have a turbidity equal to McFarland number 1 for BAP and McFarland number 0.5 for MHA. If the inoculum is too large or too small, it may affect the growth and distribution of the bacteria on the plate.

• The incubation time and temperature of the plate. The plate should be incubated aerobically at 35±2°C overnight (18 hours for MHA and 24 hours for BAP). If the incubation is too short or too long, or if the temperature is too high or too low, it may affect the growth and sensitivity of the bacteria to novobiocin.

• The measurement and interpretation of the zone size. The zone size should be measured using a ruler or a caliper from edge to edge across the diameter of the zone in millimeters. The zone size should be compared with the standard criteria for susceptibility or resistance according to CLSI guidelines. If the measurement is inaccurate or if the interpretation is incorrect, it may lead to false positive or false negative results.

Therefore, before using the novobiocin susceptibility test as a diagnostic method for S. saprophyticus, one should be aware of its limitations and follow the proper procedure and quality control measures to ensure valid and reliable results.

• Sterilize the medium properly before use, sterilize the working area, work in a sterile zone, wear proper PPE, and follow laboratory safety rules.

• Don’t pour blood in molten blood agar base if it is above 45°C.

• Pour the media into petri plates in a sterile zone before the temperature of the molten media drops below 40°C; otherwise, clumps may form.

• Ensure no air bubbles are formed while pouring the media. While leaving to solidify, place the plates in a sterile zone (inside the bio-safety cabinet) and leave the lid of the plate slightly open so that vapor can escape and there are no water drops over the solidified medium.

• Completely solidify the agar plates before use. Prepare it at least 6 hours (or 1 day) before use to ensure complete solidification. Never solidify media by placing it in the refrigerator; allow it to solidify at room temperature.

• Place the disk about 25 mm away from the edge of the plate. If using multiple antibiotic disks, place the disk at least 25 mm apart from each other.

• Incubate the plates at the correct temperature and duration as specified in the procedure.

• Measure the zone of inhibition accurately using a ruler or a caliper and compare it with the standard values.

• Use quality control strains to ensure the validity of the test results.

• Report the results as sensitive or resistant only if the test bacteria are isolated from urine samples and are coagulase-negative. Otherwise, use other methods to confirm the identity of the bacteria.

References

• Kloos WE, Schleifer KH. Simplified scheme for routine identification of human Staphylococcus species. J Clin Microbiol. 1975;1(1):82-88.

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