Novobiocin Susceptibility Test: Introduction, Principle, Procedure, Result

Novobiocin Susceptibility Test: Introduction, Principle, Procedure, Result Interpretation and Uses

Introduction of Novobiocin Susceptibility Test

In Novobiocin Susceptibility Test, the novobiocin ( antibiotic) differentiation disk is useful for presumptively distinguishing Staphylococcus saprophyticus from other coagulase-negative staphylococci (CoNS) in clinical ( urinary) specimens as shown above picture. Novobiocin is produced by the actinomycete Streptomyces nivens. Earlier it was considered a skin contaminant and only significant in immuno-compromised patients but today, Staphylococcus saprophyticus has proven to be an important uropathogen. It is second only to E. coli as the most common cause of cystitis and acute urinary tract infection (UTI) in healthy, young adult women. It tends to adhere to uroepithelial cells more often and more successfully than other staphylococcal species, this is believed to partially explain the organism’s frequent role in urinary tract infections.

Principle of Novobiocin Susceptibility Test

S. saprophyticus is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in young, middle-aged female patients and it is intrinsically resistant to novobiocin. Novobiocin is an antibiotic that interferes with DNA during DNA replication and the bacterial cell cycle. It binds to DNA gyrase and blocks adenosine triphosphatase (ATPase) activity. Screening of organisms on the basis of the following properties-Non hemolytic on blood agar, coagulase-negative, Gram reaction having Gram-positive cocci in singles, pairs, and clusters from urine cultures for novobiocin resistance is reliable presumptive identification of S. saprophyticus. The Novobiocin disk is allowed to equilibrate to room temperature. A suspension equivalent to a McFarland 0.5 opacity standard is prepared either in Tryptic Soy Broth or Sterile Water or Brain Heart Infusion (BHI) Broth using a pure 18-24 hour culture. Organisms are inoculated on a Mueller- Hinton agar plate with a sterile swab to obtain confluent growth. A disk is applied onto the inoculated agar surface and lightly press down to ensure full contact with the medium. Incubate aerobically for 18-24 hours at 35-37ºC and finally measure the diameter of the zone of inhibition around the disk, and record as susceptible or resistant. A zone of inhibition greater than 16 mm is sensitive whereas a zone of inhibition less than or equal to 16 mm is resistant.

Novobiocin Susceptibility Test Requirements

Test organisms: Gram-positive cocci in the cluster, catalase test positive, coagulase test negative, and usually nonhemolytic
Tryptic Soy Broth or Sterile Water or Brain Heart Infusion (BHI) Broth
Inoculating wire
Bunsen burner
Sterile test tubes
McFarland Densitometer ( optional)
Swab sticks
Mueller Hinton agar (MHA) plate or Blood agar
Novobiocin disk
Incubator
Control Strains: Positive control (resistant) – Staphylococcus saprophyticus (ATCC 15305) and negative control (sensitive) – Staphylococcus epidermidis (ATCC 12228)
Sliding calipers/ metric ruler

Procedure of Novobiocin susceptibility test

Allow disks to equilibrate to room temperature.
Using a pure 18-24 hour culture, prepare a suspension, equivalent to a McFarland 0.5 opacity standard, either in Tryptic Soy Broth or Sterile Water or Brain Heart Infusion (BHI) Broth.
Dip a sterile swab into the suspension and rotate it against the side of the tube above the fluid level in order to remove excess inoculum.
Inoculate Mueller Hinton Agar (MHA), Blood Agar, 5%, or Tryptic Soy Agar (TSA) plate by streaking the expressed swab over the entire agar surface and repeating in 2 planes to obtain confluent growth.
Prepare a lawn of growth over the entire plate by use of a sterile swab, swabbing over the entire plate in 3 directions and around the edge of the plate.
Allow the agar surface to dry for exactly 15 minutes before applying a disk.
Aseptically apply one novobiocin disk onto the inoculated agar surface and lightly press down to ensure full contact with the medium using sterile forceps.
Incubate the plate aerobically for 18 – 24 hours at 35 to 37°C.
Measure (in millimeters) the diameter of the zone of inhibition around the novobiocin disk using sliding calipers or a metric ruler and record it as susceptible or resistant.

Result Interpretation of Novobiocin Susceptibility Test

Sensitive – A zone of inhibition greater than 16 mm
Resistant – A zone of inhibition less than or equal to 16 mm

Uses of Novobiocin Susceptibility Test

Coagulase-negative staphylococci (CoNS) have been subdivided into two categories based on their novobiocin susceptibility test. The CoNS group that demonstrates novobiocin susceptibility includes Staphulococcus epidermidis, S. haemolyticus, S. hominis subsp. hominis, S. capitis, S. lugdunensis, S. saccharolyticus, S. warneri, and other species.
The novobiocin-resistant group consists of S. cohnii, S. xylosus and S. pulvereri.
It is useful for presumptively distinguishing Staphylococcus saprophyticus from other CoNS in clinical specimens.

Limitation of Novobiocin Susceptibility Test

The novobiocin disk is not helpful and can give misleading results if it is performed on isolates other than those from urinary samples.
Coagulase-negative staphylococci (CoNS) isolated from humans that are not Staphylococcus saprophyticus such as Staphylococcus cohnii, Staphylococcus xylosus or Staphylococcus pulvereri may also be resistant to novobiocin.
Only isolated colonies of aerobic, catalase-positive, coagulase-negative, and Gram-positive cocci in clusters are to be tested.
Biochemical, and immunological tests are further recommended for the conformation of organisms.

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