PYR Test- Objectives, Principle, Procedure, Result, Limitations

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

  1. Prior to inoculation, moisten the disk slightly with reagent-grade water.
  2. 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.
  3. Incubate at room temperature for 2 minutes.
  4. Add a drop of detector reagent, N,Ndimethylaminocinnamaldehyde.
  5. Observe for a red color within 1 minute

Result Interpretation of Pyrrolidonyl Arylamidase (PYR) Test

Result Interpretation of 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 (Spyogenes) 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,AerococcusLactococcus, most Corynebacterium (Arcanobacteriumhaemolyticum, 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

  1.  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
  2. 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.
  3. 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.

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