Pyrrolidonyl Arylamidase test, PYR - Pyrrolidonyl aminopeptidase, Principle, Uses, Procedure, Result, Interpretation

Introduction to Pyrrolidonyl Arylamidase test (PYR)

The objective of this test is to detect the presence of pyrrolidonyl arylamidase (also known as pyrrolidonyl aminopeptidase or L-pyrrolidonyl-β-naphthylamide) in microorganisms.

Principle of Pyrrolidonyl Arylamidase test (PYR)

The Pyrrolidonyl Arylamidase (PYR) test is based on the activity of L-pyrrolidonyl arylamidase (enzyme) on L-pyrrolidonyl-β-napthalamide (substrate). The enzyme hydrolyzes the substrate to produce β-napthylamine and pyroglutamic acid. The product β-napthylamine reacts with the reagent N,N-methylaminocinnamaldehyde to produce a bright red precipitate.

Principle:

L-pyrrolidonyl-β-napthalamide ==^{(L-pyrrolidonyl arylamidase)}==>β-napthylamine + pyroglutamic acid==^{(N,N-methylamino-cinnamaldehyde)}==> bright red ppt

Uses of Pyrrolidonyl Arylamidase test (PYR)

Pyrrolidonyl Arylamidase test (PYR) is used in:

• the presumptive identification of group A Streptococci (S. pyogenes).

• identify some Enterobacteriaceae (E. coli) from other gram-negative rods which are also indole-positive lactose fermenters.

• separate Enterococcus spp. from group D ß-hemolytic Streptococcus spp.

• differentiate coagulase-negative staphylococci (CoNs)- Staphylococcus haemolyticus (positive PYR) and Staphylococcus auricularis (negative PYR)

Procedure of Pyrrolidonyl Arylamidase test (PYR)

There are two methods for the detection of Pyrrolidonyl Arylamidase test (PYR) - broth method and the disc method.

Disc Method

The procedure disc method for the Pyrrolidonyl Arylamidase test (PYR) are as follows:

1. Wet the PYR disc, which has been impregnated with the substrate L-pyrrolidonyl-β-napthalamide, with 10 µl sterile distilled water or deionized water

2. Pick up 5-10 test colonies from fresh culture and smear them on the wet disc.

3. Incubate the disc at room temperature for 1-2 minutes

4. About 1-2 drops of N, N-dimethylaminocinnamaldehyde (0.01% cinnamaldehyde reagent) are added to the disc.

5. Observe for the development of red coloration within the time frame of 1-2 minutes.

Broth Method

The procedure broth method for the Pyrrolidonyl Arylamidase test (PYR) are as follows:

1. In 2-3 ml of PYR broth, 3-5 test colonies from fresh culture are inoculated

2. The mixture is incubated at 35-37°C for 4 hours

3. PYR reagent (2-3 drops) is added to the test tube

4. Development for red coloration is observed for the next 1-2 minutes

Interpretation of Pyrrolidonyl Arylamidase test (PYR)

The result of the Pyrrolidonyl Arylamidase test (PYR) can be interpreted as follows:

Positive

Cherry red/bright pink coloration developed within 1-2 minutes

Negative

No color change within the designated time. Sometimes, the blue color is observed due to a positive indole reaction.

Pyrrolidonyl Arylamidase test (PYR) positive bacteria

The list of Pyrrolidonyl Arylamidase test (PYR) positive bacteria includes:

• Group A Streptococci (Streptococcus pyogenes)

• Group D Enterococci (Enterococcus faecalis, Enterococcus faecium)

• CoNs (Staphylococcus hemolyticus, Staphylococcus lugdunensis, Staphylococcus schleiferi)

• Arcanobacterium hemolyticum (formerly known as Corynebacterium haemolyticum)

• Enterobacter

• Citrobacter

• Klebsiella

• Yersinia

• Serratia

• Aerococcus

• Gamella, Lactococcus

Pyrrolidonyl Arylamidase test (PYR) negative bacteria

The list of Pyrrolidonyl Arylamidase test (PYR) negative bacteria includes:

• Group B Streptococci (Streptococcus agalactiae)

• Streptococcus mitis

• Streptococcus bovis

• Streptococcus equinus

• Streptococcus milleri