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Species identification and simultaneous detection of rifampicin resistance in staphylococci using rpoB sequencing

Abstract number: P932

Hellmark B., Söderquist B., Unemo M.

Objectives: In recent years, coagulase-negative staphylococci (CoNS) have been recognised as causative agents of various infections, especially in immunocompromised patients and related to implanted foreign body materials. A major problem is to distinguish clinically significant CoNS strains from those representing contamination. Therefore rapid, highly sensitive and specific methods are needed for species identification and further typing. Foreign body infections caused by CoNS are difficult to treat since CoNS have the ability to produce biofilm. b-lactams and glycopeptides lack activity against biofilm producing and stationary phase bacteria. In contrast, rifampicin has the ability to penetrate biofilm. However, resistance to rifampicin may rapidly emerge and, accordingly, monotherapy should be avoided.

The aim was to develop and evaluate a rapid and objective method, based on rpoB sequencing, for species identification and simultaneous detection of rifampicin resistance in staphylococci.

Methods: Fifty-one isolates, representing 17 different Staphylococcus species, were included for species identification, and isolates (n = 30) from eight patients, obtained pre- and post-treatment with rifampicin, were included for detection of rifampicin resistance.

A real-time PCR followed by sequencing was developed for a 1052 bp segment of the rpoB gene. The sequences were analysed using BioEdit software v. 7.0.5.3 and submitted to GenBank for species identification. In case of discrepancies between the initial phenotypic species identification (using ID32Staph) and rpoB sequencing, 16S rRNA gene sequencing was used.

Results: Forty-nine (96%) of the 51 isolates were possible to species identify using rpoB sequencing and the two remaining isolates were not staphylococci according to the discrepancy analysis. Comparing the results of rpoB sequencing with the phenotypic identification revealed that eight (16%) of the 49 isolates differed regarding identified species.

Each isolate resistant to rifampicin from the eight patients displayed identical rpoB sequence as its corresponding rifampicin susceptible isolate except for one (in six patients) or two (n = 3) nonsynonymous single nucleotide polymorphisms (SNPs), or an insertion of one codon, which has not previously been described (n = 3).

Conclusion: Sequencing of the rpoB gene is a rapid, objective and accurate method for species identification and simultaneous detection of rifampicin resistance in staphylococci.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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