Glyceraldehyde-3-Phosphate Dehydrogenase-Encoding Gene as a Useful Taxonomic Tool for Staphylococcus spp

Abstract

The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific.

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

“…hominis and Staph. capitis as breast milk contaminants by using the recently described gap-RFLP method (Yugueros et al 2000(Yugueros et al , 2001, and Staph. lugdunens by 16S rRNA sequencing.…”

“…Staphylococci-specific PCR amplification of glyceraldehyde-3-phosphate dehydrogenase (gap)-encoding gene followed by restriction fragment length polymorphism (RFLP) analysis was applied to distinguish and identify staphylococci from other catalase positive cocci (Yugueros et al 2000(Yugueros et al , 2001. Catalase positive cocci, which gave no product in staphylococci-specific gap-PCR or had an unclear RFLP pattern, and all catalase negative isolates were identified by partial sequencing of 16S ribosomal RNA gene as described by Edwards et al (1989).…”

Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

“…In addition to phenotypic methods, several PCR-sequencing-based methods have been developed for the identification of Staphylococcus spp. : the 16S rRNA [4,15], hsp60 [13,26], sodA [37], rpoB [9,31], femA [51], tuf [18,30] and gap [27,53,54] genes have been used as targets. Many studies have demonstrated that genotyping methods are superior to phenotypic methods [18,28].…”

Assessment of the Public Health significance of meticillin resistant Staphylococcus aureus (MRSA) in animals and foods

“…To discriminate between staphylococcal species by means of DNA detection and sequencing polymorphisms, 16S rRNA genes are ideal (Becker, K. et al, 2004) but the hsp60 (Goh, S.H. et al, 1997), femA (Vannuffel, P. et al, 1999), sodA (Poyart, C. et al, 2001), tuf (Martineau, F. et al, 2001), rpoA (Drancourt, M. and Raoult, D., 2002), gap (Yugueros, J. et al, 2000).…”