Simultaneous Detection of Staphylococcus aureus and Coagulase-Negative Staphylococci in Positive Blood Cultures by Real-Time PCR with Two Fluorescence Resonance Energy Transfer Probe Sets

Abstract

A real-time PCR assay that uses two fluorescence resonance energy transfer probe sets and targets the tuf gene of staphylococci is described here. One probe set detects the Staphylococcus genus, whereas the other probe set is specific for Staphylococcus aureus. One hundred thirty-eight cultured isolates, which contained 41 isolates of staphylococci representing at least nine species, and 100 positive blood cultures that contained gram-positive cocci in clusters were tested. This assay was 100% sensitive and 100% specific for the detection of the Staphylococcus genus and of S. aureus.The genus Staphylococcus currently consists of 38 species (14, 15). Among staphylococci, Staphylococcus aureus is the most virulent species and the most important pathogen, but the incidence of infections caused by coagulase-negative staphylococci (CoNS) has increased throughout the world (2,28,29). S. epidermidis and other CoNS are common members of the normal flora of skin and mucous membranes and are therefore common contaminants in culture, particularly in blood cultures. A conundrum often exists in the interpretation of cultures that contain these microorganisms, since CoNS have also become an important cause of hospital-acquired infections (5, 9, 21). It is most important to distinguish between S. aureus and CoNS in clinical samples, given the differences in virulence between these organisms (2,28,29). It is also important, however, to confirm the presence of CoNS given the increasing importance of CoNS infections (27,33).Phenotypic methods have been the standard means of differentiating S. aureus from CoNS and for differentiating the members of CoNS. These methods all require growth of the organisms followed by biochemical testing, are time-consuming, and may still occasionally produce inaccurate results (7,8,23,26). A variety of genotypic methods have been explored for the rapid detection of S. aureus and/or for the detection and differentiation of CoNS. These methods include the use of traditional in situ hybridization, peptide-nucleic acid in situ hybridization, and a variety of nucleic acid amplification-based methods (1,3,11,13,17,18,22).We designed a single broad-range PCR primer set directed toward the tuf gene to amplify the most clinically relevant staphylococci. For this study, PCR products were detected by use of a LightCycler instrument (Roche Diagnostics, Indianapolis, Ind.) and two sets of fluorescence resonance energy transfer (FRET) hybridization probes. The first set of FRET hybridization probes was designed to detect a signature sequence of the Staphylococcus genus, whereas the second set of FRET hybridization probes was designed to specifically detect S. aureus. This assay can detect S. aureus and CoNS simultaneously and can differentiate S. aureus by a postamplification melt curve analysis using the F3 channel of the LightCycler system.We validated this dual-FRET-probe real-time PCR assay for Staphylococcus with lysates from 138 cultivated bacterial reference strains and well-characterized clinical isolates...

Evaluation of the LightCycler Staphylococcus M GRADE Kits on Positive Blood Cultures That Contained Gram-Positive Cocci in Clusters

“…Multiple studies have reported rapid and accurate detection of S. aureus in blood culture bottles growing GPCC, which speak to the feasibility and interest in this approach (1-3, 5, 6, 9, 10). Accurate detection of the presence of all staphylococci in blood culture bottles and simultaneous identification of S. aureus among them using realtime PCR have also been demonstrated (8). This approach identifies CoNS by actually detecting its presence rather than by assuming its presence in the absence of S. aureus in cultures growing GPCC.…”

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections

“…[20][21][22] Conventional PCR, and more recently real-time PCR, have been recognized as efficient tools for clinically applicable assays. 11,12,[23][24][25] The use of these tools is of particular interest in orthopedics, wherein the adhesion of bacteria within a biofilm on the surface of an implant may make the organism difficult to detect by conventional techniques. [15][16][17] Although some of these tools have been used to study orthopedic infections, much remains to be learned.…”

“…9,10 Clinical microbiologists are more and more commonly using the polymerase chain reaction (PCR) and other nucleic acid amplification assays for the detection and identification of microbial pathogens and to determine the most important mechanisms of antimicrobial resistance. Realtime or homogeneous PCR is a relatively new modification of the PCR, 11,12 wherein amplification and detection occur in the same reaction vessel secondary to the use of fluorescent detector molecules. In the clinical laboratory, this translates into easier to perform assays that are more rapid, features that increase the possible clinical applications of PCR assay.…”

Rapid Identification of Key Pathogens in Wound Infection by Molecular Means

“…The tuf gene encodes the elongation factor Tu, which is an essential constituent of the bacterial genome [26]. S. aureus strains produce an extracellular thermostable nuclease (TNase) with a frequency similar to that at which they produce coagulase.…”