What Is A PYR Test? – A Breakdown

A PYR Test means a Pyrrolidonyl Aminopeptidase Test and this is used to detect the presence of any pyrolidonyl arylamidase (which is also known as pyrrolidonyl aminopeptidase) activity within the Streptococcus pyogenes (also known as group A strep).

What Is A PYR Test? - A Breakdown

As well as in Enterococcus spp., some Enterbacteriaceae, and some coagulase-negative staphylococci.

This is also sometimes called PYR (L-pyrrolidonyll-β-naphthylamide) and this serves as a substrate which is used in detecting pyrrolidonyl peptidase.

A study was done by Faclam, Thacker, Fox and Eriquez, and it was reported that 98% of the group A streptococci as well as 96% of the group D enterococci hydrolyze PYR.

On top of this, the Aerococcus species is rarely isolated by a clinical laboratory, and these organisms are expected to be able to hydrolyze PYR

Facklam et al. also reported 98% of the group B streptococci, and 100% of the non-group A,  B, as well as D streptococci, 100% of the group D non-enterococci, and finally 82% of viridians have a negative PYR test result.

The Principle Of A PYR Test

PYR tests are a quick method for identification of bacteria that is based on the presence of the pyrrolidonyl arylamidase enzyme.

As well as this, the enzyme L-pyrrolidonyl arylamidase can hydrolyze the L-pyrrolidonyl-β-naphthylamide substrate which will create β-naphthylamine.

This β-naphthylamine will be detected by the presence of N, N-methylaminocinnamaldehyde reagent which will produce a noticeable red precipitate. 

After the substrate goes through hydrolysis from the peptidase, there will be a resulting b-naphthylamide which makes the aforementioned red color when the 0.01% cinnamaldehyde reagent is added.

After this, a visible inoculum of a microorganism will be rubbed on a small part of a disk that is impregnated by the substrate, and within 2 minutes hydrolysis will occur.

When this happens the cinnamaldehyde reagent will be added, and it will detect the reaction by changing its color to purple.

What Are The Uses For A PYR Test

  • PYR tests are used to get the presumptive identification of the presence of group A streptococci (known as Streptococcus pyogenes)
  • PYR tests are also used to get rapid differentiation between enterococci that are from group D ß-hemolytic streptococci.
  • It will also differentiate the presence of Staphylococci (also called positive haemolyticus which is from negative S. auricularis)
  • It can also be used to identify the presence of E.coli, and will separate it from any other indole positive, as well as lactose positive, and gram-negative rods.

What Is The Procedure Of A PYR Test

The Broth Method

  • First inoculate the PYR broth using somewhere between 3 and 5 colonies from an 18-24 hours pure culture.
  • Secondly incubate your tube aerobically at a temperature of 35 to 37 degrees Celsius for 4 hours.
  • After this add 2 to 3 drops of the PYR reagent and then observe a color change.
  • Finally look for any red color development which should happen within a couple of minutes

The Disk Method

  • First wet your PYR testing disc on your strip with deionized water or 10 µl sterile distilled water. Make sure to not flood the disc and just to wet it.
  • After this you will put between 5 and 10 colonies of your tested strain from an 18-24 hours culture onto the surface of your wetted disk using a loop to lightly smear them on.
  • Thirdly incubate this disk for 1 or 2 minutes at a room temperature.
  • After this incubation, you will add just 1 drop of N, N-dimethylaminocinnamaldehyde.
  • Finally, observe any potential red color development which should appear within a couple of minutes.

Interpreting The Results Of A PYR Test

  • If the result is positive it will be either a bright cherry-red or a bright pink within a couple of minutes, this shows the presence of Group A Streptococci, (also known by Streptococcus pyogenes), any Group D Enterococci (known as Enterococcis facecilis as well as Enterococcus faecilum), Coagulase negative Stapylococcus species like S.hemolyticus, S. schleiferi., S. lugdunensis, Citrobacter, Enterobacter, Klebsiella, Serratia, and Yersinia, as well as any Aerococcus, Lactococcus, Gamella, and most Corynebacterium (Arcanobacterium) hemolyticum.
  • If the result is negative it will have either no color change at all or go blue due to the presence of a positive indole reaction. This could show the Group B Streptococci (called Streptococcus agalactiae), Streptococcus mitis, as well as S. equinus, S. milleri, and S. bovis.
  • If there is just a pale pink color this is often just considered a weak or negative reaction.

PYR Test Quality Control

  • Positive Control: Streptococcus pyogenes (ATCC19615) and Enterococcus faecalis (ATCC29212)
  • Negative Control; Streptococcus agalactiae (ATCC10386)

The Limitations Of A PYR Test

  • A PYR test can be used for the presumptive separation of group A stretococci as well as group D enetrcocci, from any other streptococci. Any extra testing that needs a pure culture will be recommended to get a complete identification.
  • If the disk or the filter paper is too moist you can easily get a false-negative result.
  • Any false-negative results can be made if selective media or any tube biochemical agars were used for providing inocula
  • Escherichia coli as well as indole-positive Proteus can be obtained from media that contains high tryptophan content and this may lead to a blue/green color development meaning a negative result.
  • There are also some less often encountered isolates of aerococci and lactococci that could potentially be PYRase positive.
  • Any non-specific color reactions could occur if you leave reading the results for more than 20 seconds.

Frequently Asked Questions

What Are PYR Test Used For?

PYR test are utilized to test for the presence of pyrolidonyl arylamidase within the Streptococcus pyogenes (also known as group A strep), as well as in Enterococcus spp., some Enterbacteriaceae, and some coagulase-negative staphylococci.

This is sometimes called PYR, and it is used in detecting pyrrolidonyl peptidase.

What Is The Principle Of A PYR Test?

PYR tests are seen as a quick method of identifying bacteria which uses the method of detecting the presence of the pyrrolidonyl arylamidase enzyme.

What Is A PYR Test Used For?

PYR Tests are used to identify the presence of streptococci, as well as get rapid differentiation between enterococci which are from the group D ß-hemolytic streptococci.

This test also differentiates the presence of Staphylococci as well as being used to detect E.coli as well as separating it from any indole positive bacteria.

What Are The Two Methods Of Doing A PYR Test?

There are two different methods of doing a PYR Test which are the broth and the disk method. The broth method as the name suggests uses a PYR broth which is inoculated with different culture which are the incubated with a reagent added to show the results.

The disk method uses a testing disk which uses deionized water and also tests the colonies of culture after incubation.

How Do You Interpret The Results Of A PYR Test?

Both methods use a reagent to display the results with a positive result resulting in either a bright pink or cherry red color and if the result is a very weak pink or blue, or no color change the result is negative, or the test has been done wrong.

Jennifer Dawkins

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