Rapid and Accurate Species-Level Identification of Coagulase-Negative Staphylococci by Using the sodA Gene as a Target

Abstract

Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodA int ) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences. Sequence analysis revealed that the staphylococcal sodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.

Identification and detection of methicillin resistance in Non-Epidermidis coagulase-negative staphylococci

“…By conventional and commercial methods of species identification, in addition to giving low accuracy (50%-70%), are more troublesome and require more incubation time [19]. Among the isolates, 75% (178/238) were identified only by phenotypic methods and 25% (60/238) by a commercial method.…”

“…Among the isolates, 75% (178/238) were identified only by phenotypic methods and 25% (60/238) by a commercial method. Molecular methods based on the analysis of products from PCR have been developed for SCoN identification, giving improved consistency and speed [19,22]. Species that are more difficult to identify by phenotypic or commercial methods, such as S. caprae and S. equorum, were identified by PCR and sodA gene sequencing.…”

“…The samples that gave variable results in the phenotypic test identifications, or to confirm less frequent species, were run through an automated method of identification (Microscan Walkway; Dade Behringer, Deerfield, IL, USA). The automated results that gave a low percentage certainty of species identification were submitted to determination of the sodA gene by PCR amplification and sequencing with specific primers: d 1 : 5'CCITAYICITAYGAYYGCIYTIGARCC-3' and d 2 : 5'ARRTARTAIGCRTGYTCCCAIACRTC-3' [19].…”

Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

“…Analysis of specific regions of genomic DNA, on the other hand, has produced much more discriminative data. For example, several genomic targets have been effectively used for the identification of Staphylococcus species, including the 16S rRNA gene (Bialkowska-Hobrzanska et al, 1990;De Buyser et al, 1992), the tRNA gene intergenic spacer (Maes et al, 1997), the internal transcribed spacer (Couto et al, 2001), the heat-shock protein 60 (HSP60) gene (Goh et al, 1996), the chaperonin 60 gene (Goh et al, 1997), the femA gene (Vannuffel et al, 1999), the sodA gene (Poyart et al, 2001), the gap gene (Yugueros et al, 2000) and the nuc gene (Brakstad et al, 1992). Recently, an enterobacterial repetitive intergenic consensus PCR and BOX-PCR were also used in the identification of Staphylococcus epidermidis strains (Wieser & Busse, 2000).…”

Comparison phenotypic and genotypic identification of Staphylococcus species isolated from bovine mastitis

“…Coagulase-negative Staphylococcus (CoNS) have become increasingly important in human and veterinary medicine, therefore it is mandatory accurately identify the isolates to species level in order to define the clinical significance of these bacteria, to carry out a proper epidemiological surveillance, and to manage patients infected with CoNS in case of relapse (Poyart et al 2001).…”