PYR test is generally used for detecting the activity of pyrrolidonyl aryl amidase in the species of Streptococcus pyogenes which belongs to the group A family of Streptococcus and other species like Enterococcus spp, coagulase negative staphylococci and some species of Enterobacteriaceae.
This is also known as PYR (L-Pyrroliodonyl Beta-naphthylamide) that serves as a substrate foe determining the pyrroliodonyl peptidase.
Facklam, Thacker, Fox and Eriquez is reported that 98 percentage of the group A streptococci and the 96 % of the group D Enterococci hydrolyse PYR. Although the species of Aerococcus are rarely isolated in the clinical laboratory. These organisms are also expected to hydrolyse the PYR.
However, Facklam et.al reported that 98 percent of the group A streptococci and 96 percentage of the Enterococci hydrolyse the PYR. Although Aerococcus species are found very rarely isolated from the clinical laboratory and these organisms are also expected to hydrolyse PYR.
Facklam et. el later reported that there are about 98 percent of the group B Streptococci, 100 percent of the non-group A, B, D Streptococci 100 % of group D non-enterococci and the 83% of the viridians streptococci yield negative PYR test results.
PYR Test Principle
PYR is one of the rapid methods for presumptively identifying the bacteria based on their pyrrolidonyl arylamidase enzyme.
The enzyme L-pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl Beta-naphthylamine.
This Beta-naphthylamine is usually detected during the presence of the N, N-methylamino cinnamaldehyde reagent which results in the production of the bright red precipitate.
After the process of hydrolysis of the substrate with the help of peptidase, which results in the formation of a b-naphthylamide and results in the formation of a red colour upon the addition of 0.01% of the cinnamaldehyde reagent.
On rubbing a visible inoculum of the micro-organisms in a small area in the disk, that is impregnated using a substrate by hydrolysing happens within 2 minutes, during when the cinnamaldehyde reagent is added. However, the reaction is usually detected by change in colour.
PYR Test Procedure
Generally, there are about two methods used for performing the PYR test.
1. Broth PYR Test Method: Initially the PYR broth is inoculated for about 18 to 24 hours with a 3 to 5 colonies. Then the inoculated broth is incubated along with the tube aerobically at a temperature of 35 to 37ºC for about four hours, After completing the process of incubation about 2 to 3 drops of PYR reagent is added and the tube is noted for change in colour. Usually for positive test the development of the red colour within 1 to 2 minutes.
2. Disk PYR Test Method (Rapid): Disk method is one of the rapid methods used for performing the PYR test. In the process, the wet PYR test disc on the strip using distilled water for about 10µl. Instead of distilled water, deionized water can also be used. It should be noted that the disk should not be flooded while wetting. After wetting about 5 to 10 colonies of the test strain is taken from an 18 to 24-hour culture and it is placed on the surface of the disc using an inoculating loop and it is smeared lightly on it. Then the disc is incubated for about 1 to 2 minutes in a room temperature. After incubating the disc is taken out and a drop of N, N-dimethylaminocinnamaldehyde is added. After addition formation of red colour is observed within 2 minutes.
PYR Test Results
Positive PYR Test Results: If the result is positive bright pink or cherry red colour is observed within 2 minutes. For positive control, the species belonging to Group A Streptococci, Group D Enterococci, Coagulase negative Staphylococcus species such as hemolyticus, S. lugdunesis, Corynebacterium hemolyticum. Citrobacter, Aerococcus, S. schleiferi.
Negative PYR Test Results: : In case if the test is detected as negative result, then there will be no colour change. Sometimes there will be a blue colour in the medium. If the medium is appeared to be pale pink, then the reaction is considered as negative. Mostly the species belonging to Group B Streptococci, Streptococcus mitis, S. bovis, S. equis, S. milleri are tested as negative.
PYR Test Quality Control
Positive Control: Enterococcus faecalis, Streptococci’s pyogenes
Negative Control: Streptococcus agalactiae.
PYR Test Limitation
PYR test is used as a presumptive separation of the group A Streptococci and the Group D Enterococci from the other species of streptococci species.
Additional testing is done using a pure culture, and it is recommended for complete identification of the characteristics and nature of the species.
If the disk or filter paper is too moist it results in false, negative tests.
False-negative results happen if the selective medium or the biochemical agars are used for providing the inoculum.
Escherichia coli and indole positive proteus obtained from the medulla containing a high tryptophan content yields a blue-green development. This results in negative reactions.
On the other hand, some less commonly encountered species of lactococci and aerococci results in PYRase positive. In case, if the results are read after 20 seconds of performing, then it results in Non-specific colour reactions.
PYR Test Citations
- Presumptive identification of streptococci by pyrrolidonyl-beta-naphthylamide (PYR) test. Zhonghua Yi Xue Za Zhi (Taipei) . 1997 Apr;59(4):259-64.
- Value of the L-pyrrolidonyl-beta-naphthylamide hydrolysis test for identification of select gram-positive cocci. Diagn Microbiol Infect Dis . 1986 Jan;4(1):43-7.