L-pyrrolidinyl-β-naphthylamide (PYR) Test: Introduction, Principle, Test Requirements, Procedure, Result-Interpretations, and Limitations

Introduction of L-pyrrolidinyl-β-naphthylamide (PYR) Test

L-pyrrolidinyl-β-naphthylamide (PYR) test uses to identify the capability of organisms utilizing substrate, PYR by L-pyrroglutamyl amino-peptidase. Application of this test are as follows:

• Identification of Streptococcus pyogenes (PYR positive)from other beta-hemolytic Streptococci (Negative)

• Differentiation of Enterococcus species (PYR positive) from group D Streptococci (Streptococcus bovis, Streptococcus equinus) which are PYR negative.

• It is used in the identification of Escherichia coli (PYR Negative), separating it from other indoles positive, lactose positive, gram-negative rods.

• It is helpful to differentiate among the coagulase-negative Staphylococci to screen for Staphylococcus lugdunensis (PYR positive) and identify other staphylococci to the species level.

Principle of L-pyrrolidinyl-β-naphthylamide (PYR) Test

L-pyrrolidinyl-β-naphthylamide (PYR) acts as a substrate for the detection of the enzyme pyrrolidonyl peptidase produced by the organism. Following hydrolysis of the substrate by the peptidase, the resulting b-naphthylamide produces a red color upon the addition of 0.01% cinnamaldehyde reagent.

Requirements for PYR Test

• L-pyrrolidinyl-β-naphthylamide (PYR) disks
• Test organism may be of the following category

1. Catalase-negative, beta-hemolytic, gram-positive cocci with typical group A streptococcal morphology
2. Catalase-negative,gamma-oralpha-hemolytic,gram-positivecocciwithtypical enterococcal morphology
3. Oxidase-negative, indole-positive, gram-negative rods that are lactose positive on MacConkey agar, to identify E. coli
4. Coagulase-negative staphylococci, to screen for Staphylococcus lugdunensis and identify other staphylococci to the species level

• Sterile water
• Sterile sticks or inoculating loop
• Petri dish and forceps
• 0.01% cinnamaldehyde reagent
• Control strains

Positive Control (PC): Enterococcus faecalis ATCC 29212

Negative Control (NC): E. coli ATCC 25923

Procedure for PYR Disk test

1. PlaceL-pyrrolidinyl-β-naphthylamide (PYR) disk in a petri dish using forceps.
2. Put a drop of sterile water on the disk just to moisten but do not saturate.
3. Using inoculating loop or sterile stick take one or two loopful of culture from a blood agar plate that is 24 to 48 hours
4. Rub onto PYR disk.
5. Allow to react for 2 minutes (extend the time to 10 min for poorly growing organisms).
6. After the incubation period, add 1 drop of cinnamaldehyde reagent and observe for red color

Result Interpretation of L-pyrrolidinyl-β-naphthylamide (PYR) Test

 PYR test is positive: the appearance of a bright pink or cherry-red color usually within 1 min.

PYR test negative:  no color change or a blue color due to a positive indole reaction.

Note:  A pale pink reaction i.e. weak is considered PYR test negative.

Enterococcus faecalis ATCC 29212: PYR test  positive

E. coli ATCC 25923:PYR test negative

Limitations of PYR Test

1. A false-negative test can result if the disk is too moist.
2. Odd gram-positive cocci will be positive in this test but are not enterococci for such condition Gram stain is most helpful. They are generally in tetrads or clusters in the smear, are tiny colonies, or are not significant pathogens.

Further Readings

1. Cowan and Steel’s, manual for the identification of medical bacteria
2. Lynne S. Garcia, Clinical Microbiology Procedures Handbook
3. L-Pyrrolidonyl Arylamidase (PYR) Test. Procedure 13-36. Bailey & Scott’s Diagnostic Microbiology.
4. Monica Cheesbrough. District Laboratory Practice in Tropical Countries. Second Edition. Part 2. Chapter 7 Microbiological Test. 7.18.2 Streptococcus pyogenes. pp- 160.
5. Patrick R Murray. Manual of Clinical Microbiology. 8^th PRY Test. pp- 409-410.
6. PYR Disk. Remel.
7. PYR Test Kit and PYR Reagent. Hardy Diagnostics.