Species-Specific and Ubiquitous-DNA-Based Assays for Rapid Identification ofStaphylococcus aureus

Abstract

Staphylococcus aureus is the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identify S. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from an S. aureus genomic library, we have identified a chromosomal DNA fragment which is specific for S. aureus and which detected all 82 S. aureus isolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains of S. aureus isolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis of S. aureus infections.

Investigation of biofilm production and icaA and icaD genes in Staphylococcus aureus isolated from heifers and cows with mastitis

“…The S. aureus species were confirmed by PCR using the protocol and primers described by Martineau et al (1998). Bacterial DNA was extracted using the RTP ® Bacteria DNA Mini kit (Invitek, Berlin, Germany) according to manufacturer's instructions.…”

Safety-related properties of staphylococci isolated from food and food environments

“…DNA was isolated from the strains using InstaGene Matrix (Bio-Rad Laboratories, Segrate, Italy) according to the manufacturer's recommendations. RAPD profiles were obtained by PCR with the M13 primer according to the protocol of Pinto et al (2005) (Forsman et al 1997;Martineau et al 1998;Aymerich et al 2003;Morot-Bizot et al 2003 or 16S rRNA gene sequencing with the P1 and P4 primers (Klijn et al 1991). The sequencing samples were analysed with the Blast algorithm (Altschul et al 1997) to identify the species, and only strains that had a unique RAPD profile were further characterized.…”

pH value promotes growth of Staphylococcus epidermidis in platelet concentrates

“…29 Furthermore, we modified the multiplex PCR and included the amplification of the mecA gene 30 and of a DNA fragment specific to S. epidermidis (Primer Pair 2). 31 DNA amplifications were carried out with the following modified thermal cycling profile: an initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, with extension at 72°C for 2 minutes, and ending with a final extension at 72°C for 5 minutes. Furthermore, biofilm production was analyzed by the Congo red agar assay as described by Freeman and coworkers.…”