Veterynarna biotekhnolohiia – Veterinary biotechnology, 2023, 43, 175-185 [in Ukrainian]. https://doi.org/10.31073/vet_biotech43-17

SHEVCHENKO M.¹, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., TARASOV O.², e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., ANDRIICHUK A.¹, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., HONCHARENKO V.¹, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., TSARENKO T.¹, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it. 

OPTIMIZATION OF LABORATORY PCR PROTOCOLS FOR THE ACCURATE IDENTIFICATION OF S. AUREUS AND S. PSEUDINTERMEDIUS IN DOGS  

Introduction. Coagulase-positive staphylococci (CPCs) are a group of pathogenic microorganisms that can cause infections in both animals and humans. In dogs, Staphylococcus pseudintermedius is a common colonizer of healthy animals, while Staphylococcus aureus is less common in dogs. However, both of these bacteria can be zoonotic, i.e. transmitted from humans to dogs.

Distinguishing between S. aureus and S. pseudintermedius is difficult, and traditional biochemical tests may not provide accurate results.

Misidentification is particularly dangerous in the context of antibiotic resistance as understanding the source of resistant strains is crucial for effective risk management.

The goal of the work was to optimize the PCR protocol for the detection of S. aureus and S. pseudintermedius bacteria.

Materials and methods. The bacteria were obtained from two sources: museum specimens and samples collected from dogs.

The initial steps included isolation and cultivation of the bacteria. For the museum strains, they were streaked on petri dishes with agar and grown at 37°C. For samples from dogs, swabs were taken and used for inoculation on mannitol agar, followed by subcultivation to obtain pure cultures. Gram staining and enzyme tests were then performed to identify bacterial species.

DNA was extracted with the IndiSpin Pathogen Kit using DNA obtained from different dilutions of bacterial suspensions. PCR analysis was performed to amplify specific genetic markers of S. aureus and S. pseudintermedius.

To optimize the protocol, different concentrations of bacterial mass were prepared, starting with McFarland turbidity standards of 4, 2, 1, and 0.5. The lowest concentration was further diluted by serial dilutions of 10, 100 and 1000 times. To quantify these dilutions, the optical density of each was measured at 590 nm.

To ensure the accuracy of the results, the amplicons were sequenced by Sanger, and the consensus sequences were analyzed using the BLAST program.

Results of research and discussion. The optimal annealing temperature of the protocol using the S. aureus primer was within 53°C and 59°C. When DNA was isolated from a bacterial suspension in a dilution from 4 to 0.5:10 according to the McFarland standard, a septic reaction product was formed.

To optimize the protocol for the detection of Staphylococcus pseudintermedius, coagulase-positive staphylococci isolated from dogs were studied. For this protocol, it was set the annealing temperature to 56°C. To confirm the accuracy of the obtained DNA samples, Sanger sequencing was performed. This sequencing approach involved analysis of consensus sequences obtained from forward and reverse primers. BLAST alignment results of these consensus sequences showed a high degree of homology, and comparison with the NCBI reference genome revealed over 99% identity.

Conclusions and prospects for further research:

1. The protocol for species identification of S. pseudintermedius and S. aureus bacteria was developed.

2. The optimal annealing temperature was within 51–61°C for both primers. The reaction product was formed from DNA isolated from bacterial suspensions in a concentration from 4 to 0.5:10 according to the McFarland standard.

3. Sequencing of the amplicon confirmed its belonging to the S. pseudintermedius family.

The isolated DNA will be used in further studies as a confirmed positive control.

Keywords: PCR, protocol optimization, dogs, sequencing, positive control.

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