Objectives of Pyrrolidonyl Arylamidase (PYR) Test
- To determine the ability of organism to produce L-pyrrolidonyl arylamidase enzyme.
- It aids in the presumptive identification of group A streptococci (Streptococcus pyogenes) and Enterococci by the presence of the enzyme L-pyrrolidonyl arylamidase.
Principle of Pyrrolidonyl Arylamidase (PYR) Test
Pyrrolidonyl Arylamidase (PYR) test is a rapid test that is used for the presumptive identification of group A beta-hemolytic Streptococci and Enterococci based on the activity of the enzyme pyrolidonyl arylamidase. Pyrrolidonyl arylamidase (PYR), also known as pyrrolidonyl aminopeptidase, is a bacterial enzyme that hydrolyzes L-pyroglutamic acid-β-naphthylamide impregnated into the test disk that serves as the substrate for the detection of pyrolidonyl arylamidase. Hydrolysis of the substrate yields beta-naphthylamine which combines with the PYR Reagent (p-dimethylamino-cinnamaldehyde) to form a bright pink to cherry red color. The test can be performed either via the broth assay method or the rapid paper disk method.
Procedure of Pyrrolidonyl Arylamidase (PYR) Test
- Prior to inoculation, moisten the disk slightly with reagent-grade water.
- Using a wooden applicator stick, rub a small amount of several colonies of an 18 to 24 hour pure culture onto a small area of the PYR disk.
- Incubate at room temperature for 2 minutes.
- Add a drop of detector reagent, N,Ndimethylaminocinnamaldehyde.
- Observe for a red color within 1 minute
Result Interpretation of Pyrrolidonyl Arylamidase (PYR) Test
Positive test: Bright red color within 1 minute
Negative test: No color change or development of an orange or yellow color
Limitations of PYR Test
- Removing PYR test cards from the sealed bag during long-term storage may result in a decreased shelf life. Maintain cards in the sealed bag until ready to use for best performance.
- PYR may be used in the presumptive separation of group A streptococci and group D enterococci from other streptococci. Only group A streptococci and group D enterococci are PYR-positive. Other streptococci are negative; however additional testing, using a pure culture, is recommended for complete identification and may be necessary to separate group A streptococci (S. pyogenes) from beta-hemolytic enterococci.
- It is important that testing first be performed to determine that the organism is in the Streptococcus A Gram stain and catalase test should be performed to confirm the presence of gram-positive, catalase-negative cocci. The ability to hydrolyze bile esculin may be used to presumptively identify group D enterococci.
- Some Staphylococcus,Aerococcus, Lactococcus, most Corynebacterium (Arcanobacterium) haemolyticum, as well as some Enterobacteriaceae and other gram-negative bacilli, are also PYR-positive.
- A false-negative test can result if the disk or filter paper are too moist.
- Weak, pale results occur with the disk test for Staphylococcus aureus; positive results may need to be confirmed with other tests or with the tube PYR test, which is available in commercial rapid identification kits.
- False-negative tests can result if selective media or tube biochemical agars are used as inoculum for the test.
- Escherichia coli and indole-positive Proteus obtained from media containing a high tryptophan content may yield a blue-green color development which is considered as a negative result.
Quality Control of PYR Test
Enterococcus faecalis ATCC29212: positive
Streptococcus pyogenes ATCC19615: positive
Streptococcus agalactiae ATCC10386: negative
References of PYR Test
- Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
- Snyder JW, Atlas RM. Handbook of Media for Clinical Microbiology. CRC Press. Taylor & Francis Group. 6000 Broken Sound Parkway NW, Suite 300. Boca Raton, FL 33487-2742.
- Compton ST, Kania SA, Robertson AE, Lawhon SD, Jenkins SG, Westblade LF, Bemis DA. 2017. Evaluation of pyrrolidonyl arylamidase activity in Staphylococcus delphini. J Clin Microbiol 55:859–864. https://doi.org/10.1128/JCM.02076-16.