Validation of PCR-RFLP analysis of the gap gene as a useful tool for the species-level identification of staphylococcal isolates

Abstract number: p943

Grzeszczuk  S., Sabat  A., Bartoszewicz  M., Przondo-Mordarska   A., Hryniewicz  W.

Objective: 

An increase in the number of infections due to coagulate negative Staphylococcus (CoNS) has been documented. Therefore, rapid and accurate identification of CoNS species may provide important diagnostic information, which would allow the selection of an appropriate course of treatment in a timely and effective manner. The gap gene encoding glyceraldehyde-3-phosphate dehydrogenase has been proposed as a target for the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for identification of 24 Staphylococcus species. The goal of this study was to evaluate the reliability of the PCR-RFLP system in the identification of staphylococci at the species level.

Methods: 

A collection of 202 mostly clinical as well as laboratory strains comprising 30 staphylococcal species was selected. Isolates were identified to the species level by partial sequence analysis of the 16S rRNA, sodA and rpoB genes; PCR-based amplification of the gap gene followed by RFLP analysis with restriction enzyme AluI; and biochemical tests based on ID 32 STAPH (bioMerieux) and the presence of clumping factor and coagulase.

Results: 

Using the PCR-RFLP method, we examined the polymorphism of gap gene. During this study we determined the gap genotypes by AluI PCR-RFLP for further six staphylococcal species, therefore we were able to increase the list of species from 24 to 30. PCR-RFLP results were consistent with the results of partial sequencing of 16S rRNA, sodA and rpoB. Biochemical tests agreed in only 86% with other methods tested.

Conclusion: 

These results suggest that the PCR-RFLP assay provides rapid, accurate, and reliable species-level identification of staphylococci. By using a series of control strains in each experiment the assay will be easy to standardise and interpret.