Novobiocin-Susceptibility Test- Principle, Procedure and Results

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Purpose

This test is used to determine the effect of novobiocin on an organism.  Staphylococcus saprophyticus is inhibited by novobiocin; whereas S. epidermis is not.

Principle

Novobiocin is an aminocoumarin antibiotic, produced by the actinomycete Streptomyces nivens, with antibacterial property. In 1975, Kloos and Schleifer reported a simplified scheme for differentiating coagulase-negative Staphylococcus spp. which included a novobiocin disk test. Coagulase-negative staphylococci can be divided into novobiocin-susceptible and novobiocin-resistant species. Among the novobiocin-resistant species, S. saprophyticus is the one commonly recovered from humans as a cause of urinary tract infections. Therefore, screening coagulase-negative staphylococci isolated from quantitative urine cultures for susceptibility to novobiocin provides a reliable presumptive identification of this species.

Reagents

Novobiocin disks, 5 mg

 Blood agar /Mueller Hinton agar plate

Quality Control

Quality control of novobiocin test should be performed per lot/shipment date with known organisms.

Positive control: S. saprophyticus

Negative control: S. epidermidis 

Procedure

  1. Prepare a suspension of the organism to be identified in sterile distilled water or broth.
  2. The suspension should be equivalent in turbidity to a 0.5 McFarland standard.
  3. With a sterile swab, spread some of the suspension over the Blood agar /MHA plate.
  4. Aseptically place a novobiocin disk on the inoculated area. Susceptibility to furazolidone may be assessed on the same plate by placing the disks about 4 cm apart on the inoculated area. Gently tap the disk(s) with sterile forceps to assure contact with the agar surface.
  5. Incubate the plate aerobically for 18–24 hours at 35°C.

Results and Interpretation

  1. S. saprophyticus are novobiocin-resistant and will show zones of inhibition of 6 mm (no zone)–12 mm.
  2. Other coagulase-negative staphylococci and S. aureus are novobiocin-susceptible and will show zones of 16 mm or larger.
Reference

  1. Koneman’s Color Atlas and Text book of Diagnistic Microbiology.
  2. Bailey and Scott’s Diagnostic Microbiology.


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