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A novel multiplex PCR identifies 7 staphylococcal species and mecA gene directly from blood cultures

Abstract number: O27

Chatzigeorgiou K.S., Siafakas N., Tarpatzi A., Petinaki E., Zerva L.

Objectives: Besides the well-established pathogenic potential of Staphylococcus. aureus, coagulase negative species virulence is increasingly being appreciated. The aim of the present report was to describe a multiplex PCR assay suitable for the identification of 7 staphylococcal species and the detection of the methicillin resistance determinant mecA.

Methods: Eight different primer pairs targeting femA (for S. aureus, Staphylococcus hominis and Staphylococcus saprophyticus), sodA (for Staphylococcus haemolyticus and Staphylococcus capitis), fbl (for Staphylococcus lugdunensis), a gene of unknown function (for Staphylococcus epidermidis) and mecA, were used for the optimization of the multiplex PCR protocol. The assay was tested on DNA extracted from solid cultures of 213 isolates belonging to 12 staphylococcal and 17 non-staphylococcal species (6 Gram-positive and 11 Gram-negative). RFLP analysis of the tuf gene was used as a reference method for speciation of 196 staphylococcal isolates, whereas the presence of mecA gene was assayed by a uniplex PCR. The 17 non-staphylococcal strains were speciated by Phoenix System (Becton Dickinson, BD). The assay was also tested on DNA extracted directly from 21 blood culture broths (Aerobic/F, Anaerobic/F, BD), spiked with isolates previously assigned to the staphylococcal 7 species (one reference strain and two clinical from each species) included in the multiplex protocol. An organic extraction protocol using benzyl alcohol was optimized for DNA extraction from the blood culture broths.

Results: The new method identified correctly 67 strains as S. aureus, 64 as S. epidermidis, 18 as S. lugdunensis, 16 as S. haemolyticus, 10 as S. hominis, 6 as S. saprophyticus and 4 as S. capitis. No amplification products were recorded for the remaining staphylococcal species and genera. In accordance with the uniplex mecA PCR, 61 methicillin resistant staphylococci were detected by the multiplex protocol. Application of the protocol on DNA extracted directly from spiked blood bottles produced the expected results in all cases within 6–8 hours.

Conclusion: The newly developed multiplex PCR assay specifically discriminates the 7 most commonly encountered staphylococcal species and concurrently determines their resistance towards methicillin. Its application on positive blood cultures is expected to reduce significantly the turn-around time and reliable identification and susceptibility results.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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